The PMCA and Na+/Ca2+ exchange systems are two vital plasma membrane calciumpumping mechanisms in peripheral and central neurons; each of these plays a role in buffering [Ca2+]i transients via extrusion of calcium from the cytosol. PMCA, like SERCA, requires energy and pumps calcium against the chemical gradient from the cytosol to the extracellular fluid. The Na+/Ca2+ exchanger utilizes energy stored in the Na+ gradient to pump calcium out of the cellular cytosol (Werth *et al*., 1996; Blaustein & Lederer, 1999; Pottorf & Thayer, 2002; Pottorf *et al*., 2006). A noted feature of the PMCA is that its activity can be elevated by a priming stimulus which induces prolonged elevated [Ca2+]i transients. Following prolonged elevation of [Ca2+]i, the PMCA-mediated rate of recovery of depolarization-evoked [Ca2+]i is accelerated, and this elevated function can be retained for up to 40 min after the priming stimulus (Pottorf and Thayer, 2002). These studies suggest that some buffering systems are flexible and can elevate their function during times of high neuronal activity to protect the cell from [Ca2+]i overload. There are at least two mechanisms that can account for increased function of PMCA with sustained [Ca2+]i loads: induction of PMCA gene expression and interaction with calmodulin. Blocking SERCA function with SERCA antagonists combined with prolonged [Ca2+]i transients has been shown to induce PMCA gene expression (Kuo et al., 1997). Furthermore, accelerated function of PMCA with prolonged [Ca2+]i transients increased the interaction of calmodulin with PMCA (Pottorf & Thayer, 2002). Thus redundant calcium buffering components, along with functional alteration of some components mediated by molecular modulators, can compensate for a reduction in function of one or more calcium buffering components as illustrated by the case of prolonged [Ca2+]i transients. Further support for this proposition comes from studies showing that, in the failing myocardium, Na+/Ca2+ exchanger levels increase as SERCA levels decline (Hasenfuss *et al*., 1991).

Calcium Regulation in Neuronal Function with Advancing Age: Limits of Homeostasis 545

SER via SERCA pumps (Kostyuk & Verkhratsky, 1994; Verkhratsky *et al*., 1994; Shmigol *et al*., 1996). Therefore, it is clear that calcium pumps such as SERCA serve at least two functions, buffering of elevated [Ca2+]i and refilling [Ca2+]i stores to maintain calcium signaling via CICR. In CNS, SCG, and sensory neurons, SER calcium stores can be rapidly refilled by activation of voltage gated calcium channels or following depletion with caffeine they can spontaneously refill within 3–10 min via activation of store operated calcium channels (SOCC) (Friel & Tsien, 1992; Shmigol *et al*., 1994, 1996; Usachev & Thayer, 1999b;

SOCC channels have received much attention recently, and their function and modulation is an ongoing topic in excitable cell models (Cahalan, 2009). SOCC channels on the plasmalemma mediate a process called "store operated calcium entry." This is a complex process involving calcium-sensing proteins within the SER, STIM1 and STIM2, that activate SOCC. The STIM proteins contain calcium-binding sites, the "EF-hand domain", within the SER lumen and act as "sensors" of SER calcium levels. When SER calcium levels are high, STIM proteins form dimers, but when SER calcium levels fall calcium dissociates from the EF-hand domain, and the STIM proteins form oligomers and aggregate in puncta in the SER. STIM oligomers then interact with SOCC dimers to form SOCC tetramers, which conduct calcium (Cahalan, 2009). Calcium influx through SOCC coupled with SERCA-mediated

Our work with adrenergic nerve endings and isolated SCG cells has consistently shown that SERCA-mediated calcium uptake is compromised in senescent animals. As SERCA function declines with age in the SCG, we studied the impact of advancing age on refilling and release of calcium from the SER. Two protocols were used to measure rapid depolarizationevoked refilling of SER calcium stores and spontaneous refilling following caffeine-evoked depletion of SER calcium stores in isolated Fura-2 loaded SCG neurons from rats aged 6-24 months. Following caffeine-evoked depletion of SER calcium stores, rapid high K+-evoked refilling of these stores markedly declined in SCG cells from 20 and 24 month-old animals but not in 6 and 12 month-old animals. Next we measured spontaneous refilling of SER via the opening of SOCC channels and subsequent SERCA-mediated uptake of calcium into the SER following selective caffeine-evoked depletion of SER calcium stores. To ensure that the rising [Ca2+]i following caffeine-evoked depletion of SER calcium stores was mediated via activated SOCC, we used the SOCC blocker lanthanum which, indeed, abolished the influx of calcium. Spontaneous refilling of SER calcium stores significantly declined in SCG cells from 20 and 24 month-old animals. Overall, these data suggest that a functional consequence of reduced SERCA activity with advancing age is a compromise in the ability of SCG neurons to sustain release of [Ca2+]i during ongoing neuronal activity (Vanterpool *et*

In a more recent series of experiments we tested the hypothesis that reduced refilling of SER calcium stores with advancing age would reduce the contribution of CICR to electrical field stimulation (EFS)-evoked [Ca2+]i transients in isolated SCG cells from 6, 12 and 24 monthold rats (Behringer et al., 2009). We used small, graded increases of applied EFS current at a constant stimulation train length. With this protocol and measuring the peak and rate of rise of [Ca2+]i we were able to estimate the contribution of CICR to EFS-evoked elevations in [Ca2+]i. In the presence of ryanodine to block the contribution of CICR, peak and rate of rise

Baba *et al*., 2003; Vanterpool *et al*., 2005).

*al*., 2005).

calcium uptake allows for refilling of SER calcium stores.

Our studies of isolated SCG neurons lend direct support to the idea that multiple components of the calcium buffering system can compensate for a loss of SERCA function. As discussed above we found that, with advancing age, SCG neurons become more reliant on mitochondria to control high K+-evoked [Ca2+]i transients. In a study with isolated SCG neurons, when both SERCA and mitochondrial calcium uptake were blocked with thapsigargin and DNP respectively, PMCA and Na+/Ca2+ exchangers are required to control high K+-evoked [Ca2+]i transients. In this case SCG neurons from both young and old animals were able to recover from high K+-evoked [Ca2+]i transients (Tsai *et al*., 1998). These data suggest that, in peripheral adrenergic neurons, plasma membrane calcium extrusion systems can by themselves control [Ca2+]i, and their function is maintained with advancing age. In support of this study, we used a unique ATPase antagonist, vanadate, a compound that, at low concentrations (0.25 μM), selectively blocks PMCA function but does not significantly alter SERCA function. Under these conditions, the rate of recovery of [Ca2+]i transients was diminished to a greater extent in SCG neurons from old as compared to young animals (Pottorf *et al*., 2000a). This shows again that PMCA function may be elevated to meet the calcium buffering demands that occur as SERCA function declines with advancing age. Although PMCA function has been reported to decline with age in synaptosomes derived from neurons in the CNS (Qin *et al*., 1998), our studies suggest that during normal aging PMCA function in peripheral adrenergic neurons is maintained with age.

## **6. Consequences of age-related changes in SERCA function: Aging and SER calcium levels and the role of CICR in neuronal function**

Neuronal function depends in part on release of calcium from the SER in response to [Ca2+]i elevation (Usachev & Thayer, 1997, 1999a,b; Behringer *et al*., 2009). Rapid calcium buffers immediately dampen much of the calcium influx through voltage-gated calcium channels while rapid release of calcium from the SER sustains depolarization-evoked increases in [Ca2+]i. This process has been termed CICR, is mediated by RyR channels and has been shown to be relevant in modulating release of neurotransmitters and hormones. For example, when RyR are blocked and hence CICR, excitatory or inhibitory post synaptic currents, indices of presynaptic transmitter release, significantly decline (Emptage *et al*., 2001; Galante and Marty, 2003).

A unique feature of the CICR process, the "all or none" release of calcium from the SER, has been observed in peripheral sensory neurons (Usachev and Thayer, 1997). Using a combination of patch clamp and microfluorometry, [Ca2+]i was elevated with graded step depolarizations of varied time length. This method produces small graded elevations in [Ca2+]i until a "threshold" of [Ca2+]i is reached, and the full [Ca2+]i transient due to combined calcium influx and CICR is achieved. As CICR is mediated via the opening of RyR channels, agents such as caffeine, which sensitize RyR to calcium, reduce the threshold of [Ca2+] necessary to evoke CICR (Usachev and Thayer, 1997; reviewed by Usachev and Thayer, 1999a,b). In addition, ryanodine, which blocks RyR channel function can ablate CICR so that stimulation-evoked [Ca2+]i transients reflect calcium influx through calcium channels only (Usachev and Thayer, 1997; Behringer *et al*., 2009).

As CICR is a regenerative process it is sustained during ongoing neuronal activity via refilling of the SER calcium stores through calcium influx and subsequent uptake into the

Our studies of isolated SCG neurons lend direct support to the idea that multiple components of the calcium buffering system can compensate for a loss of SERCA function. As discussed above we found that, with advancing age, SCG neurons become more reliant on mitochondria to control high K+-evoked [Ca2+]i transients. In a study with isolated SCG neurons, when both SERCA and mitochondrial calcium uptake were blocked with thapsigargin and DNP respectively, PMCA and Na+/Ca2+ exchangers are required to control high K+-evoked [Ca2+]i transients. In this case SCG neurons from both young and old animals were able to recover from high K+-evoked [Ca2+]i transients (Tsai *et al*., 1998). These data suggest that, in peripheral adrenergic neurons, plasma membrane calcium extrusion systems can by themselves control [Ca2+]i, and their function is maintained with advancing age. In support of this study, we used a unique ATPase antagonist, vanadate, a compound that, at low concentrations (0.25 μM), selectively blocks PMCA function but does not significantly alter SERCA function. Under these conditions, the rate of recovery of [Ca2+]i transients was diminished to a greater extent in SCG neurons from old as compared to young animals (Pottorf *et al*., 2000a). This shows again that PMCA function may be elevated to meet the calcium buffering demands that occur as SERCA function declines with advancing age. Although PMCA function has been reported to decline with age in synaptosomes derived from neurons in the CNS (Qin *et al*., 1998), our studies suggest that during normal aging PMCA function in peripheral adrenergic neurons is maintained with

**6. Consequences of age-related changes in SERCA function: Aging and SER**

Neuronal function depends in part on release of calcium from the SER in response to [Ca2+]i elevation (Usachev & Thayer, 1997, 1999a,b; Behringer *et al*., 2009). Rapid calcium buffers immediately dampen much of the calcium influx through voltage-gated calcium channels while rapid release of calcium from the SER sustains depolarization-evoked increases in [Ca2+]i. This process has been termed CICR, is mediated by RyR channels and has been shown to be relevant in modulating release of neurotransmitters and hormones. For example, when RyR are blocked and hence CICR, excitatory or inhibitory post synaptic currents, indices of presynaptic transmitter release, significantly decline (Emptage *et al*.,

A unique feature of the CICR process, the "all or none" release of calcium from the SER, has been observed in peripheral sensory neurons (Usachev and Thayer, 1997). Using a combination of patch clamp and microfluorometry, [Ca2+]i was elevated with graded step depolarizations of varied time length. This method produces small graded elevations in [Ca2+]i until a "threshold" of [Ca2+]i is reached, and the full [Ca2+]i transient due to combined calcium influx and CICR is achieved. As CICR is mediated via the opening of RyR channels, agents such as caffeine, which sensitize RyR to calcium, reduce the threshold of [Ca2+] necessary to evoke CICR (Usachev and Thayer, 1997; reviewed by Usachev and Thayer, 1999a,b). In addition, ryanodine, which blocks RyR channel function can ablate CICR so that stimulation-evoked [Ca2+]i transients reflect calcium influx through calcium channels only

As CICR is a regenerative process it is sustained during ongoing neuronal activity via refilling of the SER calcium stores through calcium influx and subsequent uptake into the

**calcium levels and the role of CICR in neuronal function**

age.

2001; Galante and Marty, 2003).

(Usachev and Thayer, 1997; Behringer *et al*., 2009).

SER via SERCA pumps (Kostyuk & Verkhratsky, 1994; Verkhratsky *et al*., 1994; Shmigol *et al*., 1996). Therefore, it is clear that calcium pumps such as SERCA serve at least two functions, buffering of elevated [Ca2+]i and refilling [Ca2+]i stores to maintain calcium signaling via CICR. In CNS, SCG, and sensory neurons, SER calcium stores can be rapidly refilled by activation of voltage gated calcium channels or following depletion with caffeine they can spontaneously refill within 3–10 min via activation of store operated calcium channels (SOCC) (Friel & Tsien, 1992; Shmigol *et al*., 1994, 1996; Usachev & Thayer, 1999b; Baba *et al*., 2003; Vanterpool *et al*., 2005).

SOCC channels have received much attention recently, and their function and modulation is an ongoing topic in excitable cell models (Cahalan, 2009). SOCC channels on the plasmalemma mediate a process called "store operated calcium entry." This is a complex process involving calcium-sensing proteins within the SER, STIM1 and STIM2, that activate SOCC. The STIM proteins contain calcium-binding sites, the "EF-hand domain", within the SER lumen and act as "sensors" of SER calcium levels. When SER calcium levels are high, STIM proteins form dimers, but when SER calcium levels fall calcium dissociates from the EF-hand domain, and the STIM proteins form oligomers and aggregate in puncta in the SER. STIM oligomers then interact with SOCC dimers to form SOCC tetramers, which conduct calcium (Cahalan, 2009). Calcium influx through SOCC coupled with SERCA-mediated calcium uptake allows for refilling of SER calcium stores.

Our work with adrenergic nerve endings and isolated SCG cells has consistently shown that SERCA-mediated calcium uptake is compromised in senescent animals. As SERCA function declines with age in the SCG, we studied the impact of advancing age on refilling and release of calcium from the SER. Two protocols were used to measure rapid depolarizationevoked refilling of SER calcium stores and spontaneous refilling following caffeine-evoked depletion of SER calcium stores in isolated Fura-2 loaded SCG neurons from rats aged 6-24 months. Following caffeine-evoked depletion of SER calcium stores, rapid high K+-evoked refilling of these stores markedly declined in SCG cells from 20 and 24 month-old animals but not in 6 and 12 month-old animals. Next we measured spontaneous refilling of SER via the opening of SOCC channels and subsequent SERCA-mediated uptake of calcium into the SER following selective caffeine-evoked depletion of SER calcium stores. To ensure that the rising [Ca2+]i following caffeine-evoked depletion of SER calcium stores was mediated via activated SOCC, we used the SOCC blocker lanthanum which, indeed, abolished the influx of calcium. Spontaneous refilling of SER calcium stores significantly declined in SCG cells from 20 and 24 month-old animals. Overall, these data suggest that a functional consequence of reduced SERCA activity with advancing age is a compromise in the ability of SCG neurons to sustain release of [Ca2+]i during ongoing neuronal activity (Vanterpool *et al*., 2005).

In a more recent series of experiments we tested the hypothesis that reduced refilling of SER calcium stores with advancing age would reduce the contribution of CICR to electrical field stimulation (EFS)-evoked [Ca2+]i transients in isolated SCG cells from 6, 12 and 24 monthold rats (Behringer et al., 2009). We used small, graded increases of applied EFS current at a constant stimulation train length. With this protocol and measuring the peak and rate of rise of [Ca2+]i we were able to estimate the contribution of CICR to EFS-evoked elevations in [Ca2+]i. In the presence of ryanodine to block the contribution of CICR, peak and rate of rise

Calcium Regulation in Neuronal Function with Advancing Age: Limits of Homeostasis 547

participate as calcium release channels responsible for CICR (Ogawa et al., 2000). Because RyR are integral components in [Ca2+]i signaling in SCG neurons, we tested the hypothesis that, with advancing age, expression of the predominant RyR isoform(s) in adult rat SCG, along with selective modulators, is altered (Vanterpool *et al*., 2006). For this study we used F-344 rats aged 6, 12 and 24 months and molecular techniques of reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays (ELISA). ELISA was used to measure RyR protein levels as these proteins are larger than 500 kDa, limiting the

Much to our surprise we found that RyR1 mRNA was undetectable in the rat SCG, in direct contrast with other studies demonstrating that RyR1 mRNA is expressed in excitable cells, including neurons (Fill & Copello, 2002). Thus, RyR1 does not appear to play a role in mediating CICR from SER calcium stores in the SCG, regardless of animal age. However, RyR2 and RyR3 mRNA are amply present in rat SCG, appearing to be the major receptor isoforms regulating calcium release from RyR-sensitive stores in all age groups. Furthermore, RyR3 mRNA and protein levels increased from 6 to 12 months and then decreased in senescent (24 month) animals, while RyR2 mRNA and protein levels remained constant with advancing age (Vanterpool *et al*., 2006). From these data showing the impact of age on mRNA and protein levels of RyR3 it is difficult to make a straightforward conclusion. However, as discussed above, protein levels are not the only important measure

Therefore, we extended our studies to investigate possible age-related changes in selected modulators of the RyR, including phosphorylation and nNOS levels which are known to modulate levels of cADPr (Vanterpool *et al*., 2006). We found that total phosphorylation of RyR channels was not altered with age, suggesting that steady state phosphorylation of RyR and hence regulation by this mechanism does not necessarily change with advancing age. In contrast, however, nNOS protein levels increased from 6 to 12 months and significantly declined from 12 to 24 months. Since nNOS function modulates cADPr levels a reasonable inference is that cADPr levels may also decline with age. In the SCG, as there appear to be only two RyR contributing to calcium release, it is possible that the decline in RyR3 coupled with a decline in nNOS levels resulting in reduced cADPr may account in part for a decline

We have shown that caffeine-evoked release of calcium declines with age suggesting that the capacity of RyR to release calcium from the SER declines with advancing age. In addition, the contribution of CICR to EFS-evoked [Ca2+]i transients is abolished in SCG cells from senescent animals (Vanterpool *et al*., 2005; Behringer et al., 2009). Both agonist-mediated release of calcium from the SER and CICR activated via calcium influx depend on SER calcium filling levels, as well as modulation of RYR. Indeed, we have shown that SERCA function declines in the SCG (Tsai *et al*., 1998; Pottorf *et al*., 2000c). In light of these former studies a reasonable inference is that filling levels of the SER may be compromised, altering both agonist-mediated

**8. Proposed model for age-related changes in [Ca2+]i regulation in peripheral**

We have cited various reports and reviews suggesting that advancing age leads to [Ca2+]i dysregulation and neuronal loss (Choi, 1992; Kirischuk & Verkhratsky, 1996; Ichas & Mazat,

capacity of calcium release and activation of CICR from the SER.

of RyR function, as activity of these channels is be regulated by several factors.

accuracy of Western blots.

in RyR function with age.

**adrenergic neurons**

of EFS-evoked [Ca2+]i transients were significantly reduced in SCG cells from both 6 and 12 month-old animals. In contrast, in SCG cells from 24 month-old animals ryanodine had no effect on peak or rate of rise of EFS-evoked [Ca2+]i transients. These data suggest that the contribution of CICR to calcium signaling in senescent SCG neurons is virtually absent, and function of calcium signaling in senescent SCG neurons is maintained only by calcium influx through voltage gated calcium channels (Behringer *et al*., 2009). There are at least two possible mechanisms accounting for this precipitous loss of CICR in senescent neurons. The first is our demonstration of an age-related decline in SERCA function, which would result in depleted SER calcium levels with age. The second mechanism is that the function of RyR channels may also decline with age. Indeed caffeine is a ryanodine agonist and has been used to sensitize CICR to EFS-evoked [Ca2+]i transients (Usachev and Thayer, 1997). Thus, we tested the hypothesis that CICR may still possibly contribute to EFS-evoked [Ca2+]i transients in senescent neurons in the presence of the RyR agonist caffeine. Indeed, caffeine significantly increased EFS-evoked [Ca2+]i transients with short stimulation train lengths in SCG cells from 6, 12 and 24 month-old animals (Behringer *et al*., 2009). These data suggest that although CICR from the SER may not contribute to calcium signaling in senescent SCG neurons, possibly due to a combination of reduced SERCA and RyR function, senescent neurons still retain some capacity for CICR in the presence of a RyR agonist.

## **7. Altered expression of ryanodine receptors (RyR) and modulators of CICR with advancing age**

We have shown that the contribution of CICR to EFS-evoked [Ca2+]i transients is abolished with advancing age in SCG neurons; this may be a consequence of reduced SERCA and/or RyR function (Behringer et al., 2009). In this section we will discuss RyR function in more depth so as to illuminate possible age-related changes. As discussed above, release of calcium from the SER is mediated via RyR ion channels. RyR function is controlled both by gene expression and by secondary molecular modulators, which regulate the sensitivity of RyR to [Ca2+]i levels. For example, regulators of RyR play an important role in other excitable cells such as in the heart, and altered regulation of RyR appears to be a root cause of cardiac arrhythmias that occur in the elderly as well as in other pathological states (Marx and Marks, 2002). Thus, RyR are an important clinical target for therapeutic intervention in cardiovascular challenges faced by aging populations.

Modulators of RyR include FK506 binding protein, phosphorylation and intracellular molecules including cyclic adenosine diphosphate ribose (cADPr). FK506 inhibits the channel when bound to RyR, while phosphorylation of RyR reduces the binding of FK506, activating RyR by increasing its open probability (Danila and Hamilton, 2004). In the case of cADPr, in excitable cells including sympathetic neurons, this modulator can mobilize calcium from SER calcium stores and sensitize the RyR to elevated [Ca2+]i , thus reducing the threshold of elevated [Ca2+]i necessary to evoke CICR (Galione, 1993; Hua *et al*., 1994; Ogawa *et al*., 2000; Hagashida et al., 2001; Marx & Marks, 2002). Furthermore, there are reports that RyR function is influenced by neuronal nitric oxide synthase (nNOS), via nitosylation of RyR by NO itself and modulation of cADPr levels (Galione, 1993; Eu *et al*., 1999; Hagashida *et al*., 2001; Balshaw *et al*., 2002; Meissner, 2002).

Three isoforms of RyR are known; these are RyR1 (skeletal muscle), RyR2 (cardiac muscle and neurons), and RyR3 (neurons and other tissue types). All three have been shown to

of EFS-evoked [Ca2+]i transients were significantly reduced in SCG cells from both 6 and 12 month-old animals. In contrast, in SCG cells from 24 month-old animals ryanodine had no effect on peak or rate of rise of EFS-evoked [Ca2+]i transients. These data suggest that the contribution of CICR to calcium signaling in senescent SCG neurons is virtually absent, and function of calcium signaling in senescent SCG neurons is maintained only by calcium influx through voltage gated calcium channels (Behringer *et al*., 2009). There are at least two possible mechanisms accounting for this precipitous loss of CICR in senescent neurons. The first is our demonstration of an age-related decline in SERCA function, which would result in depleted SER calcium levels with age. The second mechanism is that the function of RyR channels may also decline with age. Indeed caffeine is a ryanodine agonist and has been used to sensitize CICR to EFS-evoked [Ca2+]i transients (Usachev and Thayer, 1997). Thus, we tested the hypothesis that CICR may still possibly contribute to EFS-evoked [Ca2+]i transients in senescent neurons in the presence of the RyR agonist caffeine. Indeed, caffeine significantly increased EFS-evoked [Ca2+]i transients with short stimulation train lengths in SCG cells from 6, 12 and 24 month-old animals (Behringer *et al*., 2009). These data suggest that although CICR from the SER may not contribute to calcium signaling in senescent SCG neurons, possibly due to a combination of reduced SERCA and RyR function, senescent

neurons still retain some capacity for CICR in the presence of a RyR agonist.

cardiovascular challenges faced by aging populations.

1999; Hagashida *et al*., 2001; Balshaw *et al*., 2002; Meissner, 2002).

**with advancing age**

**7. Altered expression of ryanodine receptors (RyR) and modulators of CICR**

We have shown that the contribution of CICR to EFS-evoked [Ca2+]i transients is abolished with advancing age in SCG neurons; this may be a consequence of reduced SERCA and/or RyR function (Behringer et al., 2009). In this section we will discuss RyR function in more depth so as to illuminate possible age-related changes. As discussed above, release of calcium from the SER is mediated via RyR ion channels. RyR function is controlled both by gene expression and by secondary molecular modulators, which regulate the sensitivity of RyR to [Ca2+]i levels. For example, regulators of RyR play an important role in other excitable cells such as in the heart, and altered regulation of RyR appears to be a root cause of cardiac arrhythmias that occur in the elderly as well as in other pathological states (Marx and Marks, 2002). Thus, RyR are an important clinical target for therapeutic intervention in

Modulators of RyR include FK506 binding protein, phosphorylation and intracellular molecules including cyclic adenosine diphosphate ribose (cADPr). FK506 inhibits the channel when bound to RyR, while phosphorylation of RyR reduces the binding of FK506, activating RyR by increasing its open probability (Danila and Hamilton, 2004). In the case of cADPr, in excitable cells including sympathetic neurons, this modulator can mobilize calcium from SER calcium stores and sensitize the RyR to elevated [Ca2+]i , thus reducing the threshold of elevated [Ca2+]i necessary to evoke CICR (Galione, 1993; Hua *et al*., 1994; Ogawa *et al*., 2000; Hagashida et al., 2001; Marx & Marks, 2002). Furthermore, there are reports that RyR function is influenced by neuronal nitric oxide synthase (nNOS), via nitosylation of RyR by NO itself and modulation of cADPr levels (Galione, 1993; Eu *et al*.,

Three isoforms of RyR are known; these are RyR1 (skeletal muscle), RyR2 (cardiac muscle and neurons), and RyR3 (neurons and other tissue types). All three have been shown to participate as calcium release channels responsible for CICR (Ogawa et al., 2000). Because RyR are integral components in [Ca2+]i signaling in SCG neurons, we tested the hypothesis that, with advancing age, expression of the predominant RyR isoform(s) in adult rat SCG, along with selective modulators, is altered (Vanterpool *et al*., 2006). For this study we used F-344 rats aged 6, 12 and 24 months and molecular techniques of reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays (ELISA). ELISA was used to measure RyR protein levels as these proteins are larger than 500 kDa, limiting the accuracy of Western blots.

Much to our surprise we found that RyR1 mRNA was undetectable in the rat SCG, in direct contrast with other studies demonstrating that RyR1 mRNA is expressed in excitable cells, including neurons (Fill & Copello, 2002). Thus, RyR1 does not appear to play a role in mediating CICR from SER calcium stores in the SCG, regardless of animal age. However, RyR2 and RyR3 mRNA are amply present in rat SCG, appearing to be the major receptor isoforms regulating calcium release from RyR-sensitive stores in all age groups. Furthermore, RyR3 mRNA and protein levels increased from 6 to 12 months and then decreased in senescent (24 month) animals, while RyR2 mRNA and protein levels remained constant with advancing age (Vanterpool *et al*., 2006). From these data showing the impact of age on mRNA and protein levels of RyR3 it is difficult to make a straightforward conclusion. However, as discussed above, protein levels are not the only important measure of RyR function, as activity of these channels is be regulated by several factors.

Therefore, we extended our studies to investigate possible age-related changes in selected modulators of the RyR, including phosphorylation and nNOS levels which are known to modulate levels of cADPr (Vanterpool *et al*., 2006). We found that total phosphorylation of RyR channels was not altered with age, suggesting that steady state phosphorylation of RyR and hence regulation by this mechanism does not necessarily change with advancing age. In contrast, however, nNOS protein levels increased from 6 to 12 months and significantly declined from 12 to 24 months. Since nNOS function modulates cADPr levels a reasonable inference is that cADPr levels may also decline with age. In the SCG, as there appear to be only two RyR contributing to calcium release, it is possible that the decline in RyR3 coupled with a decline in nNOS levels resulting in reduced cADPr may account in part for a decline in RyR function with age.

We have shown that caffeine-evoked release of calcium declines with age suggesting that the capacity of RyR to release calcium from the SER declines with advancing age. In addition, the contribution of CICR to EFS-evoked [Ca2+]i transients is abolished in SCG cells from senescent animals (Vanterpool *et al*., 2005; Behringer et al., 2009). Both agonist-mediated release of calcium from the SER and CICR activated via calcium influx depend on SER calcium filling levels, as well as modulation of RYR. Indeed, we have shown that SERCA function declines in the SCG (Tsai *et al*., 1998; Pottorf *et al*., 2000c). In light of these former studies a reasonable inference is that filling levels of the SER may be compromised, altering both agonist-mediated capacity of calcium release and activation of CICR from the SER.

## **8. Proposed model for age-related changes in [Ca2+]i regulation in peripheral adrenergic neurons**

We have cited various reports and reviews suggesting that advancing age leads to [Ca2+]i dysregulation and neuronal loss (Choi, 1992; Kirischuk & Verkhratsky, 1996; Ichas & Mazat,

Calcium Regulation in Neuronal Function with Advancing Age: Limits of Homeostasis 549

Given advances in molecular techniques, including the explosion of knowledge of the function of micro RNA as inhibitors of mRNA translation and hence gene function (Saugstad, 2010), future studies are limitless. These may include investigations on the impact of advancing age on the genetic expression and protein levels of multiple buffering systems, including soluble calcium-buffering proteins, SERCA, mitochondrial calcium pumps, PMCA, and the Na+/Ca2+ exchanger. Our work also strongly suggests that calcium influx through store operated calcium channels is compromised with age. Thus, genetic expression and function with advancing age of store operated channels may provide another fruitful avenue of research. Finally, all of our studies suggest that SER calcium levels decline with age, compromising CICR. New calcium indicators are now available to directly study SER calcium levels; this approach may allow more in depth studies of the impact of advancing age on SER calcium levels. These types of future studies and many more may be expected to provide more direct insight on how CICR may be altered with advancing age, potentially leading to novel therapeutic modalities to prevent and/or treat

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**10. Future directions**

**11. References**

1998; Thibault *et al*., 1998; Begley *et al*., 1999; Toescu and Verkhratsky, 2007). However, this hypothesis may be more applicable to pathological conditions as opposed to normal aging. For example, in our studies of peripheral adrenergic neurons, despite age-related changes in [Ca2+]i buffering which in turn alter [Ca2+]i signaling, the cells remain both viable and functional within reasonable limits. Thus, we pose another hypothesis: In the absence of any visible pathology, advancing age leads to subtle changes in the control of [Ca2+]i that may lead to altered neuronal function, but adjustments of other [Ca2+]i control mechanisms may allow neurons to adapt. This model is summarized in Fig. 1(B), emphasizing that loss of SERCA function may be compensated for by increased mitochondrial calcium uptake and plasma membrane calcium extrusion, so as to preserve some degree of cell viability in the face of advancing age. However, increased reliance on mitochondrial calcium buffering may have consequences for long-term viability as increased mitochondrial calcium may increase the chance of mitochondrial dysfunction and release of the apoptotic signals, caspases. This model also suggests that decline with age in SERCA function reduces SER calcium levels, thus reducing the contribution of CICR to overall calcium signaling in aging peripheral neurons.

#### **9. Summary and conclusions**

These data from our laboratory and supporting studies from others suggest that, in peripheral autonomic neurons in the absence of recognizable pathology, advancing age is a subtle and complex process that does necessarily lead to dramatic deterioration. Moreover, it is clear that advancing age does not alter the function of excitable neurons in a uniform manner, as we point out differences in the function of peripheral and central neurons with advancing age. With regards to age-related changes in [Ca2+]i regulation, we presented the idea that cell viability of peripheral neurons is maintained as they compensate for an age related decline in the function of at least one calcium-buffering system, SERCA, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemmal calcium extrusion. Increased mitochondrial calcium uptake as a compensatory mechanism may leave the cell more susceptible to apoptosis which contributes to cell death (Ichas & Mazat, 1998; Thibault *et al*.,1998; Begley *et al*., 1999; Toescu and Verkhratsky, 2007). This review summarizes the major findings in our work on the dynamics of [Ca2+]i regulation and possible consequences for autonomic nerve function with advancing age.

Based on results of our most current studies and reviews (Vanterpool *et al*., 2005, 2006; Buchholz *et al*., 2007; Behringer *et al*., 2009) and our previous work and that of others we propose the following: With advancing age an alteration in [Ca2+]i signaling and function of peripheral adrenergic neurons results from a complex interplay of mechanisms, including increased sensitivity of the NA release mechanism to calcium, decline in SERCA function that alters calcium buffering and refilling of SER calcium stores, reduced RyR3 and decline in nNOS levels, which in turn modulates cADPr levels. This combination ultimately reduces the capacity of the SER to release calcium and abolishes the CICR process. With respect to the loss of CICR, this apparently leaves calcium influx as the primary mechanism to elevate [Ca2+]i in response to nerve activation, allowing some function to continue in senescent neurons. In addition, despite loss in the contribution of CICR to EFS-evoked [Ca2+]i transients, some residual CICR capacity is still retained if RyR are sensitized to elevated [Ca2+]i with an agonist. This latter observation offers some intriguing clinical possibilities to treat an age-related alteration in calcium signaling and hence peripheral neuronal function.

## **10. Future directions**

548 Senescence

1998; Thibault *et al*., 1998; Begley *et al*., 1999; Toescu and Verkhratsky, 2007). However, this hypothesis may be more applicable to pathological conditions as opposed to normal aging. For example, in our studies of peripheral adrenergic neurons, despite age-related changes in [Ca2+]i buffering which in turn alter [Ca2+]i signaling, the cells remain both viable and functional within reasonable limits. Thus, we pose another hypothesis: In the absence of any visible pathology, advancing age leads to subtle changes in the control of [Ca2+]i that may lead to altered neuronal function, but adjustments of other [Ca2+]i control mechanisms may allow neurons to adapt. This model is summarized in Fig. 1(B), emphasizing that loss of SERCA function may be compensated for by increased mitochondrial calcium uptake and plasma membrane calcium extrusion, so as to preserve some degree of cell viability in the face of advancing age. However, increased reliance on mitochondrial calcium buffering may have consequences for long-term viability as increased mitochondrial calcium may increase the chance of mitochondrial dysfunction and release of the apoptotic signals, caspases. This model also suggests that decline with age in SERCA function reduces SER calcium levels, thus reducing the contribution of CICR to overall calcium signaling in aging peripheral

These data from our laboratory and supporting studies from others suggest that, in peripheral autonomic neurons in the absence of recognizable pathology, advancing age is a subtle and complex process that does necessarily lead to dramatic deterioration. Moreover, it is clear that advancing age does not alter the function of excitable neurons in a uniform manner, as we point out differences in the function of peripheral and central neurons with advancing age. With regards to age-related changes in [Ca2+]i regulation, we presented the idea that cell viability of peripheral neurons is maintained as they compensate for an age related decline in the function of at least one calcium-buffering system, SERCA, by increased function of other calcium-buffering systems, namely, the mitochondria and plasmalemmal calcium extrusion. Increased mitochondrial calcium uptake as a compensatory mechanism may leave the cell more susceptible to apoptosis which contributes to cell death (Ichas & Mazat, 1998; Thibault *et al*.,1998; Begley *et al*., 1999; Toescu and Verkhratsky, 2007). This review summarizes the major findings in our work on the dynamics of [Ca2+]i regulation

Based on results of our most current studies and reviews (Vanterpool *et al*., 2005, 2006; Buchholz *et al*., 2007; Behringer *et al*., 2009) and our previous work and that of others we propose the following: With advancing age an alteration in [Ca2+]i signaling and function of peripheral adrenergic neurons results from a complex interplay of mechanisms, including increased sensitivity of the NA release mechanism to calcium, decline in SERCA function that alters calcium buffering and refilling of SER calcium stores, reduced RyR3 and decline in nNOS levels, which in turn modulates cADPr levels. This combination ultimately reduces the capacity of the SER to release calcium and abolishes the CICR process. With respect to the loss of CICR, this apparently leaves calcium influx as the primary mechanism to elevate [Ca2+]i in response to nerve activation, allowing some function to continue in senescent neurons. In addition, despite loss in the contribution of CICR to EFS-evoked [Ca2+]i transients, some residual CICR capacity is still retained if RyR are sensitized to elevated [Ca2+]i with an agonist. This latter observation offers some intriguing clinical possibilities to treat an age-related alteration in calcium signaling and hence peripheral neuronal function.

and possible consequences for autonomic nerve function with advancing age.

neurons.

**9. Summary and conclusions**

Given advances in molecular techniques, including the explosion of knowledge of the function of micro RNA as inhibitors of mRNA translation and hence gene function (Saugstad, 2010), future studies are limitless. These may include investigations on the impact of advancing age on the genetic expression and protein levels of multiple buffering systems, including soluble calcium-buffering proteins, SERCA, mitochondrial calcium pumps, PMCA, and the Na+/Ca2+ exchanger. Our work also strongly suggests that calcium influx through store operated calcium channels is compromised with age. Thus, genetic expression and function with advancing age of store operated channels may provide another fruitful avenue of research. Finally, all of our studies suggest that SER calcium levels decline with age, compromising CICR. New calcium indicators are now available to directly study SER calcium levels; this approach may allow more in depth studies of the impact of advancing age on SER calcium levels. These types of future studies and many more may be expected to provide more direct insight on how CICR may be altered with advancing age, potentially leading to novel therapeutic modalities to prevent and/or treat age- and disease-related alterations in neuronal and cardiovascular function.

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**All Your Eggs in One Basket:**

*Priority Research Centre in Chemical Biology,*

*University of Newcastle*

 *Australia*

**Mechanisms of Xenobiotic Induced**

Alexander P. Sobinoff, Ilana R. Bernstein and Eileen A. McLaughlin

The irreplaceable mammalian primordial follicle represents the basic unit of female fertility, serving as the primary source of all developing oocytes in the ovary. These primordial follicles remain quiescent, often for decades, until recruited into the growing pool throughout a woman's adult reproductive years. Once recruited, <1% will reach ovulation, with the remainder undergoing an apoptotic process known as atresia (Hirshfield, 1991). Menopause, or ovarian senescence, occurs when the pool of primordial follicles becomes

Pre-mature ovarian failure (POF; or early menopause) is an ovarian defect characterised by the premature loss of menstrual cyclicity before the age of 40, well below the median age of natural menopause (51 years). Approximately 1-4% of the female population suffers from this condition, making POF a significant contributor of female infertility (Coulam et al., 1986). There is now a growing body of evidence which suggests that foreign synthetic chemicals, also known as xenobiotics, are capable of causing POF by inducing premature follicular depletion. Indeed, exposure to pesticides, workplace chemicals, chemotherapeutic agents and cigarette smoke have all been associated with primordial follicle reduction resulting in premature ovarian senescence (Hoyer and Devine, 2001; Mattison et al., 1983a,

In addition to infertility, the loss of ovarian hormones which accompanies POF has been connected with an increased risk of early morbidity and mortality (Shuster et al., 2010). With current statistics indicating an increasing trend in western women opting to delay childbirth, xenobiotic exposure could have long lasting repercussions for both the fertility and long term health of these women. In this review we discuss the susceptible nature of primordial follicles and the consequences of xenobiotic induced POF. We then examine the mechanisms of ovotoxicity for environmental toxicants and xenobiotics known to target immature follicles, and discuss the development of novel methods of wildlife fertility

**1. Introduction**

exhausted.

1983b; Sobinoff et al., 2010, 2011).

control utilizing these ovotoxicants.

**Female Reproductive Senescence**

Zhang B, Fugleholm K, Day LB, Ye S, Weller RO and Day IN (2003) Molecular pathogenesis of subarachnoid haemorrhage. *Int J Biochem Cell Biol* 35: 1341-1360. **23**

## **All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence**

Alexander P. Sobinoff, Ilana R. Bernstein and Eileen A. McLaughlin *Priority Research Centre in Chemical Biology, University of Newcastle Australia*

## **1. Introduction**

558 Senescence

Zhang B, Fugleholm K, Day LB, Ye S, Weller RO and Day IN (2003) Molecular pathogenesis

The irreplaceable mammalian primordial follicle represents the basic unit of female fertility, serving as the primary source of all developing oocytes in the ovary. These primordial follicles remain quiescent, often for decades, until recruited into the growing pool throughout a woman's adult reproductive years. Once recruited, <1% will reach ovulation, with the remainder undergoing an apoptotic process known as atresia (Hirshfield, 1991). Menopause, or ovarian senescence, occurs when the pool of primordial follicles becomes exhausted.

Pre-mature ovarian failure (POF; or early menopause) is an ovarian defect characterised by the premature loss of menstrual cyclicity before the age of 40, well below the median age of natural menopause (51 years). Approximately 1-4% of the female population suffers from this condition, making POF a significant contributor of female infertility (Coulam et al., 1986). There is now a growing body of evidence which suggests that foreign synthetic chemicals, also known as xenobiotics, are capable of causing POF by inducing premature follicular depletion. Indeed, exposure to pesticides, workplace chemicals, chemotherapeutic agents and cigarette smoke have all been associated with primordial follicle reduction resulting in premature ovarian senescence (Hoyer and Devine, 2001; Mattison et al., 1983a, 1983b; Sobinoff et al., 2010, 2011).

In addition to infertility, the loss of ovarian hormones which accompanies POF has been connected with an increased risk of early morbidity and mortality (Shuster et al., 2010). With current statistics indicating an increasing trend in western women opting to delay childbirth, xenobiotic exposure could have long lasting repercussions for both the fertility and long term health of these women. In this review we discuss the susceptible nature of primordial follicles and the consequences of xenobiotic induced POF. We then examine the mechanisms of ovotoxicity for environmental toxicants and xenobiotics known to target immature follicles, and discuss the development of novel methods of wildlife fertility control utilizing these ovotoxicants.

All Your Eggs in One Basket:

Mechanisms of Xenobiotic Induced Female Reproductive Senescence 561

cytotoxic insult (Hoyer and Devine, 2001; Mattison et al., 1983a, 1983b; Sobinoff et al., 2010, 2011). It is thought that this sensitivity may stem from the primordial follicles quiescent nature. For example, somatic cells which undergo regular rounds of proliferation constantly renew macromolecules and organelles by virtue of mitosis. However, the oocyte and granulosa cells of the primordial follicle are non-proliferative, and do not benefit from mitotic renewal, perhaps making them excessively vulnerable to xenobiotics which cause sub-lethal damage to mitochondria and other structures over time (Tarin, 1996). Similarly the location of the primordial follicle population within a poorly vascularised region of the ovarian cortex also makes them highly susceptible to toxins which damage ovarian blood vessels, with the resulting cortexual fibrosis destroying primordial follicle rich segments of

In addition to direct primordial follicle injury, certain xenobiotics which target developing follicles have been shown to cause excessive primordial follicle activation (Keating, 2009; Sobinoff et al., 2010, 2011). This may be due to a homeostatic mechanism of follicular replacement, in which destroyed developing follicles result in primordial follicle activation to replace the developing pool. If the offending xenobiotic is not removed, this could

The overall impact of xenobiotic induced follicular depletion on female reproduction depends on the type of follicle targeted for destruction, dose, and duration of exposure (Hoyer and Sipes, 1996). For example, xenobiotics which target large developing follicles have an immediately noticeable effect on female fertility. Antral follicles are the primary producers of ovarian estrogen, and therefore play an important role in the FSH-LH negative feedback loop responsible for ovulation. Xenobiotics which selectively target antral follicles consequently have harmful effects on ovarian cyclicity, effectively acting as endocrine disruptors (Jarrell et al., 1991; Mattison and Schulman, 1980). Fortunately, both prolonged and acute exposure to these ovotoxic agents only causes temporary infertility, as these follicles can be replaced by the primordial follicle pool once the harmful xenobiotic is

Conversely, xenobiotics which target small pre-antral follicles have more permanent effects on female fertility which could potentially go unrecognised for years. Due to the nonrenewing nature of primordial follicles, these xenobiotics are particularly damaging to female fertility, causing permanent infertility and premature ovarian senescence. What makes this type of ovotoxicity concerning is that it has a delayed effect on reproduction which is not made apparent until such a time that follicular recruitment cannot be supported (Hooser et al., 1994). Thus this extended period of time between cause and effect means that the detrimental action of xenobiotic contact often goes unnoticed, and consequently steps are not taken to minimise exposure until it is too late. Thus even a systemic low dose of xenobiotics may produce cumulative effects over time, resulting in the same consequences on female fertility as a large single exposure. With current statistics suggesting an increasing trend in developed countries of women opting to delay childbirth until late in their reproductive life (>30 years), accelerated follicle loss resulting from xenobiotic exposure can deprive these women of the chance to start a family in the

the ovary (Guraya, 1985; Meirow et al., 2007; van Wezel and Rodgers, 1996).

**3. Consequences of xenobiotic induced primordial follicle depletion**

potentially lead to a vicious cycle of primordial follicle depletion.

removed from the immediate environment.

conventional manner (Martin et al., 2003).

## **2. The primordial follicle: Precious and vulnerable**

Oocyte development and maturation occurs within ovarian follicles. These follicles assemble when primary oocytes (arrested at meiosis prophase I) are enveloped by a single layer of flattened granulosa cells, forming the most immature stage of follicular development, the primordial follicle. The timing of this event is species-specific, but generally occurs in the primitive ovary during foetal development (McNatty et al., 2000). Due to the nature of follicular formation, the number of oocytes established around the time of birth is finite, and represents the total number of germ cells available to the mammalian female throughout her entire life (Edson et al., 2009). It is therefore the size and persistence of this primordial follicle pool which determines the female reproductive lifespan (Fig. 1).

Fig. 1. Simple mechanistic diagram of the human female reproductive lifespan.

The first stage of folliculogenesis involves the recruitment of selected primordial follicles into the growing population. To prolong the length of the female reproductive lifespan, only a few primordial follicles are chosen for recruitment at any one time, with some follicles remaining in a quiescent (non-proliferative) state for months or years (Fig 1). This event occurs in regular waves, and is continuous from birth until ovarian senescence (McGee and Hsueh, 2000). Overall, only a few hundred of all the recruited follicles will complete folliculogenesis and undergo ovulation, with the vast majority being lost to atresia (Hirshfield, 1991). Atresia is thought to be an apoptotic process which selects the healthiest follicles for ovulation, although its mechanism of action is poorly understood. As virtually all follicles are lost during optimal follicular development, it is important that primordial follicles not only survive but also are maintained in a healthy state. Over-stimulation of primordial follicle activation and premature atresia results in the extensive depletion of the primordial follicle pool, resulting in premature ovarian senescence (Reddy et al., 2008).

There is now an increasing volume of studies which link primordial follicle depletion with xenobiotic exposure, suggesting that these irreplaceable follicles are highly sensitive to

Oocyte development and maturation occurs within ovarian follicles. These follicles assemble when primary oocytes (arrested at meiosis prophase I) are enveloped by a single layer of flattened granulosa cells, forming the most immature stage of follicular development, the primordial follicle. The timing of this event is species-specific, but generally occurs in the primitive ovary during foetal development (McNatty et al., 2000). Due to the nature of follicular formation, the number of oocytes established around the time of birth is finite, and represents the total number of germ cells available to the mammalian female throughout her entire life (Edson et al., 2009). It is therefore the size and persistence of this primordial

**2. The primordial follicle: Precious and vulnerable**

follicle pool which determines the female reproductive lifespan (Fig. 1).

Fig. 1. Simple mechanistic diagram of the human female reproductive lifespan.

The first stage of folliculogenesis involves the recruitment of selected primordial follicles into the growing population. To prolong the length of the female reproductive lifespan, only a few primordial follicles are chosen for recruitment at any one time, with some follicles remaining in a quiescent (non-proliferative) state for months or years (Fig 1). This event occurs in regular waves, and is continuous from birth until ovarian senescence (McGee and Hsueh, 2000). Overall, only a few hundred of all the recruited follicles will complete folliculogenesis and undergo ovulation, with the vast majority being lost to atresia (Hirshfield, 1991). Atresia is thought to be an apoptotic process which selects the healthiest follicles for ovulation, although its mechanism of action is poorly understood. As virtually all follicles are lost during optimal follicular development, it is important that primordial follicles not only survive but also are maintained in a healthy state. Over-stimulation of primordial follicle activation and premature atresia results in the extensive depletion of the primordial follicle pool, resulting in premature ovarian senescence (Reddy et al., 2008).

There is now an increasing volume of studies which link primordial follicle depletion with xenobiotic exposure, suggesting that these irreplaceable follicles are highly sensitive to cytotoxic insult (Hoyer and Devine, 2001; Mattison et al., 1983a, 1983b; Sobinoff et al., 2010, 2011). It is thought that this sensitivity may stem from the primordial follicles quiescent nature. For example, somatic cells which undergo regular rounds of proliferation constantly renew macromolecules and organelles by virtue of mitosis. However, the oocyte and granulosa cells of the primordial follicle are non-proliferative, and do not benefit from mitotic renewal, perhaps making them excessively vulnerable to xenobiotics which cause sub-lethal damage to mitochondria and other structures over time (Tarin, 1996). Similarly the location of the primordial follicle population within a poorly vascularised region of the ovarian cortex also makes them highly susceptible to toxins which damage ovarian blood vessels, with the resulting cortexual fibrosis destroying primordial follicle rich segments of the ovary (Guraya, 1985; Meirow et al., 2007; van Wezel and Rodgers, 1996).

In addition to direct primordial follicle injury, certain xenobiotics which target developing follicles have been shown to cause excessive primordial follicle activation (Keating, 2009; Sobinoff et al., 2010, 2011). This may be due to a homeostatic mechanism of follicular replacement, in which destroyed developing follicles result in primordial follicle activation to replace the developing pool. If the offending xenobiotic is not removed, this could potentially lead to a vicious cycle of primordial follicle depletion.

## **3. Consequences of xenobiotic induced primordial follicle depletion**

The overall impact of xenobiotic induced follicular depletion on female reproduction depends on the type of follicle targeted for destruction, dose, and duration of exposure (Hoyer and Sipes, 1996). For example, xenobiotics which target large developing follicles have an immediately noticeable effect on female fertility. Antral follicles are the primary producers of ovarian estrogen, and therefore play an important role in the FSH-LH negative feedback loop responsible for ovulation. Xenobiotics which selectively target antral follicles consequently have harmful effects on ovarian cyclicity, effectively acting as endocrine disruptors (Jarrell et al., 1991; Mattison and Schulman, 1980). Fortunately, both prolonged and acute exposure to these ovotoxic agents only causes temporary infertility, as these follicles can be replaced by the primordial follicle pool once the harmful xenobiotic is removed from the immediate environment.

Conversely, xenobiotics which target small pre-antral follicles have more permanent effects on female fertility which could potentially go unrecognised for years. Due to the nonrenewing nature of primordial follicles, these xenobiotics are particularly damaging to female fertility, causing permanent infertility and premature ovarian senescence. What makes this type of ovotoxicity concerning is that it has a delayed effect on reproduction which is not made apparent until such a time that follicular recruitment cannot be supported (Hooser et al., 1994). Thus this extended period of time between cause and effect means that the detrimental action of xenobiotic contact often goes unnoticed, and consequently steps are not taken to minimise exposure until it is too late. Thus even a systemic low dose of xenobiotics may produce cumulative effects over time, resulting in the same consequences on female fertility as a large single exposure. With current statistics suggesting an increasing trend in developed countries of women opting to delay childbirth until late in their reproductive life (>30 years), accelerated follicle loss resulting from xenobiotic exposure can deprive these women of the chance to start a family in the conventional manner (Martin et al., 2003).

All Your Eggs in One Basket:

exposure.

Mechanisms of Xenobiotic Induced Female Reproductive Senescence 563

of a three tiered enzymatic detoxification mechanism (Conney, 1982). Unfortunately, this oxygenation often results in the bioactivation of the parent xenobiotic into a more reactive and therefore toxic metabolite (Harrigan et al., 2004; Melendez-Colon et al., 1999). Indeed, many of Ahr's known xenobiotic ligands, such as the polycyclic aromatic hydrocarbons benzo[a]pyrene (BaP), 9:10-dimethyl-1:2-benzanthracene (DMBA), and 3-methylcholanthrene (3-MC), cause primordial follicle destruction through Ahr initiated cytochrome P450 induced bioactivation (Borman et al., 2000; Mattison and Thorgeirsson, 1979). For example, BaP is initially metabolised by Ahr regulated cyp1A1 and cyp1B1 enzymes resulting in its biotransformation into 7,8-diol, and 9,10-diol macromolecular-adduct forming metabolites within the ovary. Inhibition of Ahr by -naphthoflavone nullifies its effects on primordial follicle destruction (Bengtsson et al., 1983; Mattison et al., 1983a).

Fig. 2. Molecular mechanism of Arh-Arnt directed gene expression in response to xenobiotic

In addition to detoxification, the development of Ahr-deficient mice has revealed a physiological role for Ahr in regulating reproduction, growth and development (Benedict et al., 2000, 2003; Nebert et al., 1984; Robles et al., 2000; Schmidt et al., 1996). Benedict et al.

In addition to permanent infertility, the loss of ovarian hormones which accompanies early menopause has been associated with an increased risk for a variety of health problems. For example, estrogen deficiency (a consequence of menopause) is the most common cause of osteoporosis in humans (Cenci et al., 2003). Bone loss results from the absence of estrogen production by maturing ovarian follicles, which leads to a subsequent increase in FSH production due to the negative feedback of estrogen on pituitary gonadotropin secretion. In terms of bone remodelling, increased FSH production stimulates tumor necrosis factor (TNF) secretion, which in turn increases osteoclast formation and bone reabsorption (Cenci et al., 2003). Menopause induced estrogen withdrawal has also been associated with an increase in many traditional cardiovascular risk factors, including body fat redistribution, insulin resistance and high blood pressure, increased plasma triglyceride levels and highdensity lipid cholesterol absorption (Bilianou, 2008; Rosano et al., 2007). Increased risk for Alzheimer's disease is also associated with the menopause induced loss of sex steroid hormones as evidenced by various epidemiological and experimental studies, although some clinical findings refute this evidence (Pike et al., 2009).

Over the course of the 20th century, the average life expectancy for women in the developing world has increased by ~40%, resulting in women now living up to a third of their lives in post menopausal years. Unfortunately, this means that women are now spending a larger proportion of their life with increased health risks brought about by the onset of menopause. In addition, increased risk resulting from xenobiotic induced premature menopause means an enhanced chance for problems. It is therefore important to understand the mechanisms behind xenobiotic induced primordial follicle depletion.

## **4. Mechanisms of xenobiotic induced primordial follicle depletion**

#### **4.1 The Aryl Hydrocarbon Receptor**

The Aryl Hydrocarbon Receptor (Ahr) is a ligand activated transcription factor implicated in the regulation of a variety of physiological and developmental effects, including xenobiotic metabolism, cell cycle progression, apoptosis and oxidative stress (Denison and Heath-Pagliuso, 1998; Nebert et al., 2000). In its inactivated state, Ahr is found in the cytoplasm bound to a number of molecular chaperones including hsp90, Xap2, and p23 (Carlson and Perdew, 2002; Petrulis and Perdew, 2002). Ligand binding causes conformational changes which expose a nuclear import signal on the Ahr, resulting in its translocation into the nucleus (Pollenz et al., 1994). Once imported the Ahr-ligand receptor complex disassociates with its chaperones and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription factor with high affinity to specific DNA sequences known as xenobiotic-response elements (XRE) within the promoter region of a variety of genes, inducing transcription (Fig.2) (Reyes et al., 1992).

The Ahr-ARNT ligand activated transcription factor is known to regulate the toxicity of various xenobiotic compounds such as polycyclic aromatic hydrocarbons, polychlorinated dibenzofurans and polychlorinated biphenyls which are found ubiquitously in the environment and are highly resistant to metabolic breakdown (Nguyen and Bradfield, 2007; Stapleton and Baker, 2003). In an adaptive response to their accumulation in the cell, Ahr induces the expression of a number of xenobiotic metabolising enzymes, including members of the cytochrome P450 A and B families which oxygenate the intruding xenobiotic as part

In addition to permanent infertility, the loss of ovarian hormones which accompanies early menopause has been associated with an increased risk for a variety of health problems. For example, estrogen deficiency (a consequence of menopause) is the most common cause of osteoporosis in humans (Cenci et al., 2003). Bone loss results from the absence of estrogen production by maturing ovarian follicles, which leads to a subsequent increase in FSH production due to the negative feedback of estrogen on pituitary gonadotropin secretion. In terms of bone remodelling, increased FSH production stimulates tumor necrosis factor (TNF) secretion, which in turn increases osteoclast formation and bone reabsorption (Cenci et al., 2003). Menopause induced estrogen withdrawal has also been associated with an increase in many traditional cardiovascular risk factors, including body fat redistribution, insulin resistance and high blood pressure, increased plasma triglyceride levels and highdensity lipid cholesterol absorption (Bilianou, 2008; Rosano et al., 2007). Increased risk for Alzheimer's disease is also associated with the menopause induced loss of sex steroid hormones as evidenced by various epidemiological and experimental studies, although

Over the course of the 20th century, the average life expectancy for women in the developing world has increased by ~40%, resulting in women now living up to a third of their lives in post menopausal years. Unfortunately, this means that women are now spending a larger proportion of their life with increased health risks brought about by the onset of menopause. In addition, increased risk resulting from xenobiotic induced premature menopause means an enhanced chance for problems. It is therefore important to understand the mechanisms

The Aryl Hydrocarbon Receptor (Ahr) is a ligand activated transcription factor implicated in the regulation of a variety of physiological and developmental effects, including xenobiotic metabolism, cell cycle progression, apoptosis and oxidative stress (Denison and Heath-Pagliuso, 1998; Nebert et al., 2000). In its inactivated state, Ahr is found in the cytoplasm bound to a number of molecular chaperones including hsp90, Xap2, and p23 (Carlson and Perdew, 2002; Petrulis and Perdew, 2002). Ligand binding causes conformational changes which expose a nuclear import signal on the Ahr, resulting in its translocation into the nucleus (Pollenz et al., 1994). Once imported the Ahr-ligand receptor complex disassociates with its chaperones and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription factor with high affinity to specific DNA sequences known as xenobiotic-response elements (XRE) within the promoter region of a variety of

The Ahr-ARNT ligand activated transcription factor is known to regulate the toxicity of various xenobiotic compounds such as polycyclic aromatic hydrocarbons, polychlorinated dibenzofurans and polychlorinated biphenyls which are found ubiquitously in the environment and are highly resistant to metabolic breakdown (Nguyen and Bradfield, 2007; Stapleton and Baker, 2003). In an adaptive response to their accumulation in the cell, Ahr induces the expression of a number of xenobiotic metabolising enzymes, including members of the cytochrome P450 A and B families which oxygenate the intruding xenobiotic as part

**4. Mechanisms of xenobiotic induced primordial follicle depletion**

some clinical findings refute this evidence (Pike et al., 2009).

behind xenobiotic induced primordial follicle depletion.

genes, inducing transcription (Fig.2) (Reyes et al., 1992).

**4.1 The Aryl Hydrocarbon Receptor**

of a three tiered enzymatic detoxification mechanism (Conney, 1982). Unfortunately, this oxygenation often results in the bioactivation of the parent xenobiotic into a more reactive and therefore toxic metabolite (Harrigan et al., 2004; Melendez-Colon et al., 1999). Indeed, many of Ahr's known xenobiotic ligands, such as the polycyclic aromatic hydrocarbons benzo[a]pyrene (BaP), 9:10-dimethyl-1:2-benzanthracene (DMBA), and 3-methylcholanthrene (3-MC), cause primordial follicle destruction through Ahr initiated cytochrome P450 induced bioactivation (Borman et al., 2000; Mattison and Thorgeirsson, 1979). For example, BaP is initially metabolised by Ahr regulated cyp1A1 and cyp1B1 enzymes resulting in its biotransformation into 7,8-diol, and 9,10-diol macromolecular-adduct forming metabolites within the ovary. Inhibition of Ahr by -naphthoflavone nullifies its effects on primordial follicle destruction (Bengtsson et al., 1983; Mattison et al., 1983a).

Fig. 2. Molecular mechanism of Arh-Arnt directed gene expression in response to xenobiotic exposure.

In addition to detoxification, the development of Ahr-deficient mice has revealed a physiological role for Ahr in regulating reproduction, growth and development (Benedict et al., 2000, 2003; Nebert et al., 1984; Robles et al., 2000; Schmidt et al., 1996). Benedict et al.

All Your Eggs in One Basket:

localised bioactivation.

Rajapaksa et al., 2007b).

Mechanisms of Xenobiotic Induced Female Reproductive Senescence 565

neighbouring somatic ovarian cells and taken up by the primordial oocyte, contributing to

A number of studies performed *in vitro* have revealed that the ovary is capable of the localised bioactivation of a number of xenobiotics into ovotoxic intermediates which target primordial follicles for destruction (Rajapaksa et al., 2007a, 2007b). An example of this localised bioactivation is the reported metabolism of the polycyclic aromatic hydrocarbon DMBA (Fig.3). Ovarian exposure to DMBA disrupts folliculogenesis, resulting in the destruction of all follicle populations leading to POF in rodents, although recent evidence suggests an alternate mechanism of ovotoxicity resulting in primordial follicle depletion in the mouse (Mattison and Schulman, 1980; Sobinoff et al., 2011). This toxicity has been attributed to the bioactivation of DMBA into its ultimate DNA-adduct forming intermediate DMBA-3,4-diol-1,2-epoxide (Shiromizu and Mattison, 1985). DMBA is bioactivated by Cyp1B1 to a 3,4-epoxide which is then converted into a 3,4-diol by the microsomal epoxide hydrolase (MeH) phase II enzyme. This intermediate is then further modified by either Cyp1A1 or Cyp1B1 to form the ultimate ovotoxicant DMBA-3,4-diol-1,2-epoxide (Shimada and Fujii Kuriyama, 2004; Shimada et al., 2001). These three enzymes required for DMBA's biotransformation are all expressed and induced by DMBA exposure in the murine ovary (Igawa et al., 2009; Rajapaksa et al., 2007b; Shimada et al., 2003). In further support of localised DMBA bioactivation, inhibition of MeH in cultured rat ovaries inhibited DMBA induced ovotoxicity, while ovarian culture in the presence of DMBA-3,4-diol induced significantly more primordial follicle depletion than DMBA alone (Igawa et al., 2009;

Fig. 3. Metabolism of DMBA and VCD into their ovotoxic metabolites.

Another example of localised bioactivation is the conversion of the industrial chemical 4- Vinylcyclohexene (VCH) into VCH diepoxide (VCD) (Fig. 3). VCH is metabolised by cytochrome P450 phase I enzymes to form VCM-monoepoxide (VCM), which is then converted into VCD. Studies have shown VCD to be the ultimate ovotoxicant, targeting both primordial and primary follicles for depletion (Hu et al., 2001; Smith et al., 1990; Sobinoff et al., 2010). As demonstrated *in vivo* and *in vitro* via knockout studies, VCH/VCM is bioactivated into VCD exclusively by the cyp2e1 isoform in the ovary (Rajapaksa et al.,

(2000) demonstrated that ovaries from mice deficient for Ahr expression contained significantly more fully formed primordial follicles compared to wild type mice on PND2-3. Robles et al (2000) found similar results, identifying more than a two-fold significant increase in the primordial follicle pool of Ahr deficient PND4 mice compared to wild type mice. These results suggest a developmental role for Ahr in regulating primordial follicle formation and atresia in the mouse. Although the exact details of Ahr role in the regulation of the primordial follicle pool have yet to be determined, given that Ahr xenobiotic ligands cause primordial follicle depletion, we hypothesise that part of these ovotoxic compounds method of ovotoxicity may involve perturbed AhR developmental signalling, inducing premature primordial follicle atresia.

#### **4.2 Bioactivation**

Humans come into contact with a variety of xenobiotics over the course of their lifetime, and have evolved a number of physiological mechanisms designed to remove their harmful influence from within the body. Hydrophilic xenobiotics tend to be less toxic, as the body is able to directly excrete them relatively unchanged. However, if the xenobiotic is lipophilic, it will need to be modified by a series of biochemical reactions before it can be eliminated (Pavek and Dvorak, 2008). This series of biochemical reactions is termed biotransformation, and can be divided into two phases. Phase I metabolism involves the introduction or exposure of a reactive polar group on the xenobiotic via oxidation, resulting in a more reactive/water soluble metabolite to facilitate excretion and/or the induction of phase II metabolism. The cytochrome p450 super family of oxidases catalyse the majority of these reactions, although other oxidases, esterases, amidases, and monooxygenases can also be involved (Schroer et al., 2010). Phase II metabolism involves the conjugation of charged species such as glutathione, sulphate, glycine or glucuronic acid to the phase I metabolite to increase its water solubility (Kohalmy and Vrzal, 2011). The addition of these large anionic groups detoxifies reactive electrophiles, resulting in a more polar metabolite which can be actively transported out of the cell. These reactions are carried out by a broad range of transferases, such as glutathione S-transferase, UDP-glucuronosyltransferases, sulfotransferases, N-acetyltransferases, and methyltransferases (Jancovaa et al., 2010).

Unfortunately, phase I metabolism of xenobiotics by the liver and other tissues occasionally results in the production of a more cytotoxic metabolite, a process known as bioactivation (Dekant, 2009). These highly reactive metabolites are electrophilic, and are capable of forming covalent bonds (or adducts) with the nucleophilic centers of cellular macromolecules, such as proteins, DNA, and RNA. Cellular toxicity occurs when these adducts disrupt the normal structure and/or function of these macromolecules, resulting in apoptosis, necrosis or carcinogenesis. The main site of xenobiotic biotransformation within the body is the liver, although the ovary is capable of both phase I and phase II metabolism (Igawa et al., 2009; Rajapaksa et al., 2007a, 2007b; Shimada et al., 2003). Therefore, there is potential for the vulnerable primordial follicle to come into contact with bioactivated ovotoxic metabolites via several routes of exposure. Bioactivated metabolites produced by the liver maybe stable enough to diffuse back into the venous circulatory system, resulting in direct ovarian exposure. Additionally, as the primordial follicle is capable of expressing xenobiotic metabolising enzymes itself, oocytes may be exposed to localised bioactivation. Finally, the xenobiotic may be bioactivated locally into its ovotoxic metabolite by

(2000) demonstrated that ovaries from mice deficient for Ahr expression contained significantly more fully formed primordial follicles compared to wild type mice on PND2-3. Robles et al (2000) found similar results, identifying more than a two-fold significant increase in the primordial follicle pool of Ahr deficient PND4 mice compared to wild type mice. These results suggest a developmental role for Ahr in regulating primordial follicle formation and atresia in the mouse. Although the exact details of Ahr role in the regulation of the primordial follicle pool have yet to be determined, given that Ahr xenobiotic ligands cause primordial follicle depletion, we hypothesise that part of these ovotoxic compounds method of ovotoxicity may involve perturbed AhR developmental signalling, inducing

Humans come into contact with a variety of xenobiotics over the course of their lifetime, and have evolved a number of physiological mechanisms designed to remove their harmful influence from within the body. Hydrophilic xenobiotics tend to be less toxic, as the body is able to directly excrete them relatively unchanged. However, if the xenobiotic is lipophilic, it will need to be modified by a series of biochemical reactions before it can be eliminated (Pavek and Dvorak, 2008). This series of biochemical reactions is termed biotransformation, and can be divided into two phases. Phase I metabolism involves the introduction or exposure of a reactive polar group on the xenobiotic via oxidation, resulting in a more reactive/water soluble metabolite to facilitate excretion and/or the induction of phase II metabolism. The cytochrome p450 super family of oxidases catalyse the majority of these reactions, although other oxidases, esterases, amidases, and monooxygenases can also be involved (Schroer et al., 2010). Phase II metabolism involves the conjugation of charged species such as glutathione, sulphate, glycine or glucuronic acid to the phase I metabolite to increase its water solubility (Kohalmy and Vrzal, 2011). The addition of these large anionic groups detoxifies reactive electrophiles, resulting in a more polar metabolite which can be actively transported out of the cell. These reactions are carried out by a broad range of transferases, such as glutathione S-transferase, UDP-glucuronosyltransferases, sulfotransferases, N-acetyltransferases, and methyltransferases (Jancovaa et al., 2010).

Unfortunately, phase I metabolism of xenobiotics by the liver and other tissues occasionally results in the production of a more cytotoxic metabolite, a process known as bioactivation (Dekant, 2009). These highly reactive metabolites are electrophilic, and are capable of forming covalent bonds (or adducts) with the nucleophilic centers of cellular macromolecules, such as proteins, DNA, and RNA. Cellular toxicity occurs when these adducts disrupt the normal structure and/or function of these macromolecules, resulting in apoptosis, necrosis or carcinogenesis. The main site of xenobiotic biotransformation within the body is the liver, although the ovary is capable of both phase I and phase II metabolism (Igawa et al., 2009; Rajapaksa et al., 2007a, 2007b; Shimada et al., 2003). Therefore, there is potential for the vulnerable primordial follicle to come into contact with bioactivated ovotoxic metabolites via several routes of exposure. Bioactivated metabolites produced by the liver maybe stable enough to diffuse back into the venous circulatory system, resulting in direct ovarian exposure. Additionally, as the primordial follicle is capable of expressing xenobiotic metabolising enzymes itself, oocytes may be exposed to localised bioactivation. Finally, the xenobiotic may be bioactivated locally into its ovotoxic metabolite by

premature primordial follicle atresia.

**4.2 Bioactivation**

neighbouring somatic ovarian cells and taken up by the primordial oocyte, contributing to localised bioactivation.

A number of studies performed *in vitro* have revealed that the ovary is capable of the localised bioactivation of a number of xenobiotics into ovotoxic intermediates which target primordial follicles for destruction (Rajapaksa et al., 2007a, 2007b). An example of this localised bioactivation is the reported metabolism of the polycyclic aromatic hydrocarbon DMBA (Fig.3). Ovarian exposure to DMBA disrupts folliculogenesis, resulting in the destruction of all follicle populations leading to POF in rodents, although recent evidence suggests an alternate mechanism of ovotoxicity resulting in primordial follicle depletion in the mouse (Mattison and Schulman, 1980; Sobinoff et al., 2011). This toxicity has been attributed to the bioactivation of DMBA into its ultimate DNA-adduct forming intermediate DMBA-3,4-diol-1,2-epoxide (Shiromizu and Mattison, 1985). DMBA is bioactivated by Cyp1B1 to a 3,4-epoxide which is then converted into a 3,4-diol by the microsomal epoxide hydrolase (MeH) phase II enzyme. This intermediate is then further modified by either Cyp1A1 or Cyp1B1 to form the ultimate ovotoxicant DMBA-3,4-diol-1,2-epoxide (Shimada and Fujii Kuriyama, 2004; Shimada et al., 2001). These three enzymes required for DMBA's biotransformation are all expressed and induced by DMBA exposure in the murine ovary (Igawa et al., 2009; Rajapaksa et al., 2007b; Shimada et al., 2003). In further support of localised DMBA bioactivation, inhibition of MeH in cultured rat ovaries inhibited DMBA induced ovotoxicity, while ovarian culture in the presence of DMBA-3,4-diol induced significantly more primordial follicle depletion than DMBA alone (Igawa et al., 2009; Rajapaksa et al., 2007b).

Fig. 3. Metabolism of DMBA and VCD into their ovotoxic metabolites.

Another example of localised bioactivation is the conversion of the industrial chemical 4- Vinylcyclohexene (VCH) into VCH diepoxide (VCD) (Fig. 3). VCH is metabolised by cytochrome P450 phase I enzymes to form VCM-monoepoxide (VCM), which is then converted into VCD. Studies have shown VCD to be the ultimate ovotoxicant, targeting both primordial and primary follicles for depletion (Hu et al., 2001; Smith et al., 1990; Sobinoff et al., 2010). As demonstrated *in vivo* and *in vitro* via knockout studies, VCH/VCM is bioactivated into VCD exclusively by the cyp2e1 isoform in the ovary (Rajapaksa et al.,

All Your Eggs in One Basket:

O2

(TUNEL) analysis (Klaunig and Kamendulis, 2004).

Mechanisms of Xenobiotic Induced Female Reproductive Senescence 567

hydroxyguanine, and Terminal deoxynucleotidyl transferase dUTP nick end labelling

Fig. 4. Biochemical pathways outlining the mechanisms of xenobiotic induced ROS production which may contribute to primordial follicle depletion. Abbreviations: Fe, iron;

Another mechanism of xenobiotic induced ROS formation is the phase I bioactivation of the offending xenobiotic into reactive and redox active o-quinone metabolites. As mentioned previously, the PAH BaP is converted into 7,8-diol, and 9,10-diol by Ahr induced cyp1A1 and cyp1B1 enzymes in the ovary (Bengtsson et al., 1983). Studies have also shown that cyp1A1 is also capable of converting BaP into the BaP o-quinones benzo[a]pyrene-3,6-dione and benzo[a]pyrene-6,12-dion (Schwarz et al., 2001). Additionally, cyp 1A1 bioactivated BaP 7,8-diol can be further metabolised via NAD(P)+-dependent oxidation by the phase I dihydrodiol dehydrogenase Akr1c1 enzyme, resulting in the formation of a ketol. This ketol then undergoes tautomerisation to form catechol 7,8-dihydroxybenzo[a]pyrene. Two subsequent 1-electron auto-oxidation events produce a *o*-semiquinone anion, followed by the formation of the o-quinone benzo[a]pyrene-7,8-dione (Trevor et al., 1996). Given the increases observed in cyp 1A1 expression in the ovary in response to BaP exposure, and the relatively high level of dihydrodiol dehydrogenase expression in the ovary compared to the

, superoxide; H2O2, hydrogen peroxide; HO, hydroxyl radical.

2007a). Rajapaksa et al (2007a) cultured neonatal ovaries from both cyp2e1+/+ and cyp2e1-/ neonatal mice in VCM and VCD containing media. Both VCH metabolites caused primordial follicle depletion in cyp2e1+/+ cultured ovaries. However, unlike VCD, VCM did not produce an ovotoxic affect in cyp2e1-/- cultured ovaries, thus demonstrating its role in VCH induced bioactivation.

## **4.3 Xenobiotic induced reactive oxygen species generation**

Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and the highly toxic hydroxyl free radical, are highly reactive oxygen-containing molecules which are produced naturally as a consequence of oxidative energy metabolism (Valko et al., 2007). These short lived ROS play an important role in regulating signal transduction, selectively oxidizing cysteine residues on proteins resulting in a variety of reversible molecular interactions (Janssen-Heininger et al., 2008). However, in excess these highly unstable molecules may lead to perturbed signal transduction and/or oxidative damage to cellular macromolecules, inducing DNA mutations, lipid peroxidation and premature protein degradation. These molecular lesions coupled with perturbed signal transduction can ultimately result in abnormal cellular function, apoptosis and necrosis (Valko et al., 2006, 2007; Wells et al., 2009).

The ovary is a highly redox sensitive organ, with oocytes themselves being particularly vulnerable to excess ROS exposure due to the low rates of oxidative repair in post-mitotic cells (Cadenas and Davies, 2000; Terman et al., 2006). According to the free radical hypothesis of ageing, non-renewing primordial follicles, which can remain quiescent for many years, gradually produce ROS through electron leakage from the mitochondrial electron transport chain (Tarin, 1996). Over time this excess ROS damages the mitochondrial membranes, leading to more electron leakage and further ROS production. Given the redox sensitive nature of primordial follicles, it is reasonable to assume that the generation of xenobiotic induced ROS formed through detoxification may exacerbate this process, contributing to primordial follicle loss (Bondy and Naderi, 1994; Danielson, 2002; Wells et al., 2009).

Xenobiotic enhanced ROS formation may occur via several mechanisms in the primordial follicle (Fig.4). If the ovotoxic xenobiotic contains a quinone-like structure, it may undergo redox cycling with the corresponding semiquinone radical to produce superoxide anions. Further enzymatic and/or spontaneous dismutation of the superoxide anions produces hydrogen peroxide, which can further react with trace amounts of iron or other transition metals to form hydroxyl free radicals (Bolton et al., 2000). Given the futile cyclical nature of redox cycling, this would allow a relatively small concentration of quinone-like xenobiotics to generate an amplified production of ROS in the ovary (Park et al., 2005). For example, menadione (MEN), a synthetic vitamin K with a quinone-like structure, is a potent toxicant which exerts its cytotoxic affect via quinone cycling (Thor et al., 1982). Recently, we examined the effects of MEN on folliculogenesis in neonatal mouse ovaries *in vitro* (Sobinoff et al., 2010). This study found that MEN caused wide spread oxidative stress and DNA damage resulting in primordial and small developing follicle destruction, as evidenced by the detection of increased levels of the hydroxyl radical–induced mutagenic DNA lesion 8-

2007a). Rajapaksa et al (2007a) cultured neonatal ovaries from both cyp2e1+/+ and cyp2e1-/ neonatal mice in VCM and VCD containing media. Both VCH metabolites caused primordial follicle depletion in cyp2e1+/+ cultured ovaries. However, unlike VCD, VCM did not produce an ovotoxic affect in cyp2e1-/- cultured ovaries, thus demonstrating its role in

Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and the highly toxic hydroxyl free radical, are highly reactive oxygen-containing molecules which are produced naturally as a consequence of oxidative energy metabolism (Valko et al., 2007). These short lived ROS play an important role in regulating signal transduction, selectively oxidizing cysteine residues on proteins resulting in a variety of reversible molecular interactions (Janssen-Heininger et al., 2008). However, in excess these highly unstable molecules may lead to perturbed signal transduction and/or oxidative damage to cellular macromolecules, inducing DNA mutations, lipid peroxidation and premature protein degradation. These molecular lesions coupled with perturbed signal transduction can ultimately result in abnormal cellular function, apoptosis and necrosis (Valko et al., 2006,

The ovary is a highly redox sensitive organ, with oocytes themselves being particularly vulnerable to excess ROS exposure due to the low rates of oxidative repair in post-mitotic cells (Cadenas and Davies, 2000; Terman et al., 2006). According to the free radical hypothesis of ageing, non-renewing primordial follicles, which can remain quiescent for many years, gradually produce ROS through electron leakage from the mitochondrial electron transport chain (Tarin, 1996). Over time this excess ROS damages the mitochondrial membranes, leading to more electron leakage and further ROS production. Given the redox sensitive nature of primordial follicles, it is reasonable to assume that the generation of xenobiotic induced ROS formed through detoxification may exacerbate this process, contributing to primordial follicle loss (Bondy and Naderi, 1994; Danielson, 2002; Wells et

Xenobiotic enhanced ROS formation may occur via several mechanisms in the primordial follicle (Fig.4). If the ovotoxic xenobiotic contains a quinone-like structure, it may undergo redox cycling with the corresponding semiquinone radical to produce superoxide anions. Further enzymatic and/or spontaneous dismutation of the superoxide anions produces hydrogen peroxide, which can further react with trace amounts of iron or other transition metals to form hydroxyl free radicals (Bolton et al., 2000). Given the futile cyclical nature of redox cycling, this would allow a relatively small concentration of quinone-like xenobiotics to generate an amplified production of ROS in the ovary (Park et al., 2005). For example, menadione (MEN), a synthetic vitamin K with a quinone-like structure, is a potent toxicant which exerts its cytotoxic affect via quinone cycling (Thor et al., 1982). Recently, we examined the effects of MEN on folliculogenesis in neonatal mouse ovaries *in vitro* (Sobinoff et al., 2010). This study found that MEN caused wide spread oxidative stress and DNA damage resulting in primordial and small developing follicle destruction, as evidenced by the detection of increased levels of the hydroxyl radical–induced mutagenic DNA lesion 8-

**4.3 Xenobiotic induced reactive oxygen species generation**

VCH induced bioactivation.

2007; Wells et al., 2009).

al., 2009).

hydroxyguanine, and Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) analysis (Klaunig and Kamendulis, 2004).

Fig. 4. Biochemical pathways outlining the mechanisms of xenobiotic induced ROS production which may contribute to primordial follicle depletion. Abbreviations: Fe, iron; O2 , superoxide; H2O2, hydrogen peroxide; HO, hydroxyl radical.

Another mechanism of xenobiotic induced ROS formation is the phase I bioactivation of the offending xenobiotic into reactive and redox active o-quinone metabolites. As mentioned previously, the PAH BaP is converted into 7,8-diol, and 9,10-diol by Ahr induced cyp1A1 and cyp1B1 enzymes in the ovary (Bengtsson et al., 1983). Studies have also shown that cyp1A1 is also capable of converting BaP into the BaP o-quinones benzo[a]pyrene-3,6-dione and benzo[a]pyrene-6,12-dion (Schwarz et al., 2001). Additionally, cyp 1A1 bioactivated BaP 7,8-diol can be further metabolised via NAD(P)+-dependent oxidation by the phase I dihydrodiol dehydrogenase Akr1c1 enzyme, resulting in the formation of a ketol. This ketol then undergoes tautomerisation to form catechol 7,8-dihydroxybenzo[a]pyrene. Two subsequent 1-electron auto-oxidation events produce a *o*-semiquinone anion, followed by the formation of the o-quinone benzo[a]pyrene-7,8-dione (Trevor et al., 1996). Given the increases observed in cyp 1A1 expression in the ovary in response to BaP exposure, and the relatively high level of dihydrodiol dehydrogenase expression in the ovary compared to the

All Your Eggs in One Basket:

depletion (Devine et al., 2004).

**4.4 Xenobiotic induced primordial follicle activation**

depletion in cultured rat ovaries (Vlahos et al., 1994).

Mechanisms of Xenobiotic Induced Female Reproductive Senescence 569

administration (Bhattacharya and Keating, 2011). Additionally, rodents given the same dose of VCD over a period of several days caused specific primordial follicle depletion only after 15 days of continual dosing (Springer et al., 1996). The significant decrease in GSH concentrations almost immediately after exposure, coupled with the delayed loss of follicles following chronic exposure suggest that GSH reduction over time due to VCD detoxification could leave the susceptible primordial follicle vulnerable to increasing concentrations of xenobiotic induced ROS, resulting in primordial follicle destruction. It is pertinent that a single dose of a higher concentration of VCD (320 mg/kg) causes significant primordial follicle depletion 6 days after exposure, but is not specific to the primordial follicle pool (Devine et al., 2004). Additionally, VCD *in vitro* culture assays have linked an increase in Gstp expression with the first signs of primordial follicle loss after 6 days of exposure in neonatal rat ovaries (Bhattacharya and Keating, 2011; Keating et al., 2010). As Gstp catalyses VCD-GSH conjugation, the increase in enzymatic expression and therefore activity could have contributed to the observed primordial follicle loss due to a reduction in GSH/GSSG oxidative buffer. Conversely, substituting VCD culture media with antioxidant such as GSH does not prevent primordial follicle depletion, suggesting it is not the ultimate cause of

Traditionally, studies attempting to identify the molecular mechanisms behind xenobiotic induced POF have focused on premature follicular atresia as the main source of primordial follicle depletion. However, there is now a growing body of evidence which suggests that xenobiotics cause primordial follicle depletion through accelerated primordial follicle activation (Keating, 2009, 2011; Sobinoff et al., 2010, 2011). A study of VCD and MXC induced primordial follicle depletion has revealed a selective mechanism of pre-antral ovotoxicity involving small developing follicle atresia and primordial follicle activation both *in vitro* and *in vivo* (Sobinoff et al., 2010). Extracted neonatal mouse ovaries cultured in either VCD or MXC were immunopositive for the apoptotic markers caspase 2, caspase 3, and TUNEL in small developing follicles from the primary stage onward, but were absent in primordial follicles (Fig.5). In addition, the primordial follicles in VCD and MXC cultured ovaries expressed proliferating cell nuclear antigen (PCNA), a marker of primordial follicle activation (Picut et al., 2008; Tománek and Chronowska, 2006). VCD and MXC exposure also induces primordial follicle activation and developing follicle atresia *in vivo* as evidenced by increased primordial follicle PCNA expression and histomorphological analysis (Sobinoff et al., 2010). Microarray analysis confirmed via qPCR also showed VCD and MXC upregulated PI3K/Akt and mTOR signalling, two synergistic pathways intimately associated with primordial follicle activation (Reddy et al., 2010). Further evidence for PI3K/Akt signalling in VCD induced primordial follicle activation comes from a study conducted by Hoyer et al (2009), in which LY294002, an inhibitor of PI3K, prevented primordial follicle

The polycyclic aromatic hydrocarbon DMBA, which was previously thought to cause indiscriminate follicular destruction, has also been shown to cause pre-antral ovotoxicity through selective immature follicle destruction and primordial follicle activation (Mattison and Schulman, 1980; Sobinoff et al., 2011). In addition to showing signs of maturing follicle atresia (caspase 2, caspase 3, TUNEL) and primordial follicle activation (PCNA), DMBA

liver, it is reasonable to assume BaP may be exerting part of its ovotoxic affect through oquinone formation (Hou et al., 1994). Indeed, both benzo[a]pyrene-3,6-dione and benzo[a]pyrene-6,12-dion were detected in rat ovaries after a single dose exposure of BaP in rodents (Ramesh et al., 2010).

The un-natural "uncoupling" of phase I cytochrome P450 enzymes may also contribute to xenobiotic induced ovotoxicity via ROS production. Cytochrome P450 enzymes use H+ obtained from NADPH to reduce O2, which leads to the production of hydrogen peroxide and/or superoxide anion radicals as part of phase I oxygenation. Unfortunately, the P450 catalytic cycle can be uncoupled, resulting in the release of the reactive hydrogen peroxide and/or superoxide anion radical from the enzyme substrate complex (Meunier et al., 2004). Although all cytochrome P450 enzymes experience uncoupling, cyp 2E1 experiences a high rate of the phenomenon (Caro and Cederbaum, 2004). Even in the absence of substrate, cyp 2E1 undergoes un-natural "uncoupling" due to its NADPH oxidase activity independent of phase I metabolism (Ekstrom and Ingelman-Sundberg, 1989). As described previously, VCH is exclusively bioactivated by cyp 2E1 to produce the ovotoxic metabolite VCD. It is therefore possible that VCH may partially cause primordial follicle depletion via excess ROS production. Indeed, studies conducted in our laboratory have demonstrated VCD itself, along with the pesticide methoxychlor (MXC) and MEN, is capable of inducing cyp 2E1 expression and oxidative stress in the form of 8-hydroxyguanine adduct formation in primordial follicles (Sobinoff et al., 2010).

Another mechanism by which ovotoxic xenobiotics may cause oxidative stress is through the depletion of glutathione peroxidase (GSH) via detoxification. GSH is the body's most abundant antioxidant, providing protection against all forms of oxidative stress by scavenging ROS by virtue of its reducing thiol group, forming oxidised glutathione disulfide (GSSG) (Kidd, 1997). The glutathione system (GSH/GSSG ratio) acts as a homoeostatic redox buffer that contributes to maintenance of the cellular redox balance, with a reduction in the GSH/GSSG ratio indicating oxidative stress (Schafer and Buettner, 2001). In addition to its function as a ROS scavenger, GSH is also employed in the phase II metabolism of many ovotoxic xenobiotics (Keating et al., 2010; Tsai-Turton et al., 2007; Wu and Berger, 2008). For example, VCD is conjugated to GSH by the glutathione S-transferase Gst isoform pi (Gstp) as part of phase II detoxification in the ovary (Keating et al., 2010).

The mammalian ovary itself is highly redox sensitive, with maturing oocytes containing the highest concentration of GSH compared to any other cell type in the body (Calvin et al., 1986; Clague et al., 1992; Luderer et al., 2001). It is therefore likely that ovarian somatic and germ cell GSH plays an important role in protecting ovarian follicles from damage by ovotoxic xenobiotics. This is especially evident in primordial follicles, where a natural decrease in the GSH/GSSG ratio with advancing reproductive age increases primordial follicle susceptibility to xenobiotic induced destruction (Mattison et al., 1983b). Therefore, we hypothesise that the detoxification of ovotoxic xenobiotics via GSH conjugation reduces the GSH/GSSG ratio in primordial follicles, leaving them vulnerable to oxidative stress and primordial follicle depletion. Indeed, DMBA detoxification involves GSH conjugation, and its ovotoxic ROS production can be reduced through the addition of GSH, curbing its ovotoxicity (Tsai-Turton et al., 2007). There is controversial evidence for this mechanism of ovotoxicity in VCD induced primordial follicle loss. Rodent exposure to VCD was shown to reduce GSH concentrations by 25% and 55% in rat and mouse ovaries 2 hours after VCD

liver, it is reasonable to assume BaP may be exerting part of its ovotoxic affect through oquinone formation (Hou et al., 1994). Indeed, both benzo[a]pyrene-3,6-dione and benzo[a]pyrene-6,12-dion were detected in rat ovaries after a single dose exposure of BaP in