**2.1 Sildenafil attenuates left ventricular dysfunction in ischemic heart failure**

Salloum et al. showed that chronic treatment with sildenafil preserves cardiomyocytes post AMI through reduction of myocardial necrosis, apoptosis and hypertrophy thereby limiting the progression of HF (Salloum et al., 2008a). This study used a murine model of post-MI remodeling by permanent ligation of the left coronary artery. The experimental protocol is illustrated in Figure 1. LV function was assessed at 7 and 28 days post MI. Cardiac function

Fig. 1. Experimental protocol illustrating various parameters studied at different time points

suboptimal due to endothelial dysfunction [damaged NO synthase (NOS)], and recently myocardial PDE-5 expression has been shown to increase in patients with heart failure (Pokreisz et al., 2009). In this regard, targeting PDE-5 is a promising therapeutic approach

A number of pioneering investigations from our laboratory have demonstrated that PDE-5 inhibitors attenuate ischemic injury in animal and cell models (Ockaili et al., 2002; Salloum et al., 2003). In animal models, sildenafil and vardenafil exerted an infarct-sparing effect when given before ischemia (Ockaili et al., 2002; Salloum et al., 2003; Salloum et al., 2006) or at the time of reperfusion (Salloum et al., 2007). Furthermore, chronic treatment with sildenafil immediately after permanent occlusion of the left descending coronary artery (LAD) in mice attenuated ischemic cardiomyopathy (Salloum et al., 2008a). These cardioprotective effects are mediated by activation of protein kinase G (PKG), increased expression of endothelial and inducible nitric oxide synthase (eNOS & iNOS), and augmented Bcl-2/Bax ratio. Due to their powerful anti-ischemic effects, PDE-5 inhibitors became promising candidates for the preservation of cardiac function following AMI. In fact, several studies demonstrated that PDE-5 inhibition preserved left ventricular (LV)

**2. PDE-5 Inhibitors preserve myocardial function following infarction**

**2.1 Sildenafil attenuates left ventricular dysfunction in ischemic heart failure**

Salloum et al. showed that chronic treatment with sildenafil preserves cardiomyocytes post AMI through reduction of myocardial necrosis, apoptosis and hypertrophy thereby limiting the progression of HF (Salloum et al., 2008a). This study used a murine model of post-MI remodeling by permanent ligation of the left coronary artery. The experimental protocol is illustrated in Figure 1. LV function was assessed at 7 and 28 days post MI. Cardiac function

> **Sildenafil (0.71 mg/kg; ip; BID) or Saline (volume matched; ip; BID)**

**Week 1 Week 2 Week 3 Week 4**

**Daily monitoring: Pain, appetite/drinking, behavior and responsiveness**

Fig. 1. Experimental protocol illustrating various parameters studied at different time points

•**Echocardiography** •**Body Weight** •**Heart weight** •**Lung weight** •**TUNEL** •**Survival**

for treatment of cardiovascular disease and dysfunction.

function in failing hearts as discussed in the following sections.

•**Echocardiography** •**Body Weight** •**Heart weight** •**Lung weight** •**TUNEL** •**Survival**

**LAD Occlusion or Sham Surgery**

**Baseline Echo**

**8 week-old ICR Mice**

> **24 hr Infarct Size**

was evaluated by echocardiography using the Vevo770TM imaging system (VisualSonics, Inc., Toronto, Canada). A 30-MHz probe was utilized to obtain two-dimensional, M-mode and Doppler imaging from parasternal short-axis view at the level of the papillary muscles and the apical four-chamber view (Schiller et al., 1989). M-mode images of the LV were obtained and systolic and diastolic wall thickness (anterior and posterior) and LV endsystolic and end-diastolic diameters (LVESD and LVEDD, respectively) were measured.

Figure 2 is representative of M-mode images from mice on day 28 post MI. The hearts from sham and sildenafil-treated mice exhibited a smaller LV cavity and thicker infarct wall compared to the saline-treated mice. Increase in LVEDD, LVESD and a decrease in anterior wall diastolic thickness (AWDT), anterior wall systolic thickness (AWST) and fractional shortening (FS) in saline- and sildenafil-treated mice (vs. baseline and sham) were observed on day 7 and 28.

Fig. 2. M-mode images from mouse LV treated with sildenafil or vehicle 28 days after MI

Sildenafil-treated mice had smaller LVEDD, LVESD, greater FS, and lower Tei index (reflecting better myocardial performance) on day 7 and 28 as compared to saline-treated group (P<0.05, Figure 3). Sildenafil-treated animals also had a shorter isovolumetric relaxation time (reflective of lower LV end-diastolic pressure) 28 days after AMI when compared to saline-treated animals (11±3 vs. 27±7 ms, respectively, P=0.03), which was not different from sham operated animals (10±3, P=NS). AWDT and AWST were also greater in sildenafil-treated animals (vs. saline-treated animals, P<0.05) on day 7 and 28 post MI showing a protective effect in the peri-infarct region, while no differences in PWDT and PWST were seen. Aneurysmatic dilatation of the anterior wall and apex was observed on day 28 in 90% of saline-treated mice and 62% of sildenafil treated animals (P>0.05).

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 101

Fig. 4. Tadalafil preserves LV function at 24 hours following MI through PKG and H2S

**2.3 Sildenafil treatment 3 days following MI mitigates the progression of heart failure**  In the previous studies, PDE-5 inhibitors were administered either shortly prior to or immediately after infarction. This raises the question whether the preservation of cardiac function observed was a true phenomenon, or it was simply secondary to the anti-infarct effect of these drugs. Specially, little is known about the effects of PDE-5 inhibition on limiting adverse remodeling independent of its ability to modulate infarct size. This concept is clinically relevant, particularly in patients with advanced ischemic HF, because necrosis

signaling

Moreover, the number of aneurysmatic segments [based on a 16-segment map (Schiller et al., 1989)] was 2.9 in saline-treated animals vs. 1.1 in sildenafil-treated animals (P<0.05).

Fig. 3. Echocardiography results of LV function at 28 days post-MI in sildenafil- and salinetreated mice

#### **2.2 Tadalafil preserves left ventricular function following MI through PKG-dependent generation of hydrogen sulfide**

We also studied the effect of a longer acting PDE-5 inhibitor, tadalafil, on cardiac function in an acute model of myocardial infarction (Salloum et al., 2009). After baseline transthoracic echocardiography, adult male mice were injected i.p. with vehicle (10% DMSO) or tadalafil (1 mg/kg) with or without KT5823 (KT, PKG blocker, 1 mg/kg) or dl-propargylglycine [PAG, Cystathionine-γ-lyase (CSE, H2S-producing enzyme) blocker; 50 mg/kg] 1 h prior to coronary artery ligation for 30 min and reperfusion for 24 h, whereas C57BL-wild type and CSEknockout mice were treated with either vehicle or tadalafil. After reperfusion, repeat echocardiography was performed. Similar to sildenafil, tadalafil preserved cardiac performance following MI as compared to vehicle. In this study, since ischemia was limited to 30 minutes, none of the groups presented with significant LV dilatation at 24 h post infarction, however, tadalafil preserved fractional shortening (FS: 31±1.5%) compared to control (FS: 22±4.8%, P<0.05, Figure 4). Baseline FS was 44±1.7%. KT and PAG abrogated the preservation of LV function with tadalafil by a decline in FS to 17±1% and 23±3%, respectively.

Moreover, the number of aneurysmatic segments [based on a 16-segment map (Schiller et al., 1989)] was 2.9 in saline-treated animals vs. 1.1 in sildenafil-treated animals (P<0.05).

**AWDT (mm)**

**A**

**0.00 0.15 0.30 0.45 0.60 0.75 0.90**

**0.25 0.50 0.75 1.00 1.25 1.50**

**Tei**

**Saline (N=6) Sildenafil (N=6) Sham (N=6)**

Fig. 3. Echocardiography results of LV function at 28 days post-MI in sildenafil- and saline-

**2.2 Tadalafil preserves left ventricular function following MI through PKG-dependent**

We also studied the effect of a longer acting PDE-5 inhibitor, tadalafil, on cardiac function in an acute model of myocardial infarction (Salloum et al., 2009). After baseline transthoracic echocardiography, adult male mice were injected i.p. with vehicle (10% DMSO) or tadalafil (1 mg/kg) with or without KT5823 (KT, PKG blocker, 1 mg/kg) or dl-propargylglycine [PAG, Cystathionine-γ-lyase (CSE, H2S-producing enzyme) blocker; 50 mg/kg] 1 h prior to coronary artery ligation for 30 min and reperfusion for 24 h, whereas C57BL-wild type and CSEknockout mice were treated with either vehicle or tadalafil. After reperfusion, repeat echocardiography was performed. Similar to sildenafil, tadalafil preserved cardiac performance following MI as compared to vehicle. In this study, since ischemia was limited to 30 minutes, none of the groups presented with significant LV dilatation at 24 h post infarction, however, tadalafil preserved fractional shortening (FS: 31±1.5%) compared to control (FS: 22±4.8%, P<0.05, Figure 4). Baseline FS was 44±1.7%. KT and PAG abrogated the preservation

of LV function with tadalafil by a decline in FS to 17±1% and 23±3%, respectively.

**0.1 0.2 0.3 0.4 0.5 0.6**

**index**

**C**

**B**

**AWST (mm)**

**baseline 7 days 28 days sham**

**P=0.21 P=0.017**

**P=0.14 P=0.001**

**P=0.019 P=0.032**

**D**

**F**

**E**

**0.0 baseline 7 days 28 days sham**

**0.00 baseline 7 days 28 dayssham**

**baseline 7 days 28 days sham**

**P=0.003 P<0.001**

**P=0.011 P=0.007**

**P<0.001**

**0.0 baseline 7 days 28 days sham**

**0.0 baseline 7 days 28 days sham**

**P=0.003**

**LVEDD (mm)**

**0.0 1.0 2.0 3.0 4.0 5.0 6.0**

**1.0 2.0 3.0 4.0 5.0 6.0**

**FS (%)**

treated mice

**LVESD (mm)**

**10.0 20.0 30.0 40.0 50.0 60.0**

**generation of hydrogen sulfide**

Fig. 4. Tadalafil preserves LV function at 24 hours following MI through PKG and H2S signaling

#### **2.3 Sildenafil treatment 3 days following MI mitigates the progression of heart failure**

In the previous studies, PDE-5 inhibitors were administered either shortly prior to or immediately after infarction. This raises the question whether the preservation of cardiac function observed was a true phenomenon, or it was simply secondary to the anti-infarct effect of these drugs. Specially, little is known about the effects of PDE-5 inhibition on limiting adverse remodeling independent of its ability to modulate infarct size. This concept is clinically relevant, particularly in patients with advanced ischemic HF, because necrosis

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 103

protective effects against ischemia/reperfusion injury in the infant rabbit hearts as well. In this study, we used the model of coronary artery occlusion and reperfusion in infant rabbits (Bremer et al., 2005), which is similar to our previously described adult rabbit model of myocardial infarction. The benefits of this work are immense since this model may be applicable in pediatrics, and especially in pediatric cardiovascular surgery where there may be periods of ischemia/reperfusion injury. Also, we used two-dimensional (2D) and Doppler trans-esophageal echocardiography (TEE) for the estimation of LV cardiac output (LVCO) and aortic velocity time integral (VTI) in this model. A 10-Fr AcuNav diagnostic ultrasound probe (Acuson Corp., Siemens, Iselin, NJ) was inserted into the esophagus, at baseline, after the 30 min period of ischemia, and after 3 h of reperfusion in both the control and sildenafil groups. Standardized 2D imaging in a long axis view of the LV to show LV inflow across the mitral valve and LV outflow tract (LVOT) was obtained. Aortic flow Doppler across the aortic valve was performed in a long axis view of the LV and LVOT to obtain LVCO. The standard equation: mean velocity (cm/s) × flow area (cm2) × 60 (s/min), where mean velocity (cm/s) = VTI (in cm/beat) ÷ RR interval (s/beat), was used to obtain LVCO, expressed in milliliters per minute. Laminar Doppler flow across the aortic valve confirmed the absence of aortic stenosis. Color Doppler assessment was made of both the aortic and mitral valves again in the long axis view at baseline, after the ischemic period, and after 3 h of reperfusion for the presence or absence of mitral or aortic regurgitation. Subjective functional assessment was also made after ischemia and reperfusion to demonstrate at least left ventricular apical diminished contractility to confirm infarction. In this study, we showed that both the control and sildenafil-treated groups had comparable LVCO and aortic VTI at baseline. The controls had a decline in LVCO and aortic VTI immediately after the 30-min period of ischemia (28% and 27% lower than baseline values, respectively, p < 0.05), whereas the LVCO and aortic VTI increased in the sildenafil group after ischemia (43% and 45% higher than baseline values, respectively, n = 6 per group, p < 0.05). Both groups, however, had significant decline in LVCO after 3 h of reperfusion (54% of baseline in the sildenafil group, p < 0.05, and 62% of baseline in the control group, p < 0.05), and were not statistically significantly different from each other (n = 4–6 per group). Both groups demonstrated a decrease in aortic VTI after 3 h of reperfusion. However, this decline was only statistically significant in the control group compared with baseline values. None of the rabbits had aortic stenosis or developed aortic regurgitation for the duration of the study. Moreover, both the control and sildenafil groups demonstrated a comparable amount of mitral regurgitation (no more than mild) after ischemia/reperfusion, and none of

the rabbits had baseline mitral regurgitation.

**2.5 Sildenafil and vardenafil preserve LV function in female mice**

Since the impact of PDE-5 inhibitors on the female cardiovascular system following ischemia remains unknown, we interrogated the effect of sildenafil and vardenafil on ischemia/reperfusion injury in female mice. In this study, adult female mice were pretreated (ip, bid) with sildenafil (0.7 mg/kg), vardenafil (0.14 mg/kg) or saline one hour before left coronary artery ligation for 30 minutes and reperfusion for 24 hours (Salloum et al., 2008b). Cardiac function, evaluated using echocardiography, showed that LV enddiastolic and end-systolic diameters increased 7 days post myocardial infarction with saline

has a negligible role in a post-infarct setting (Anversa et al., 1993). To address this question, we administered sildenafil 3 days following MI (Chau et al., 2011). Specifically, we sought to determine if sildenafil treatment following LV dysfunction, defined as FS less than 25% at day 3 post-MI, could prevent the progression of HF in a permanent LAD occlusion model. At 3 days post MI, mice receiving sildenafil or saline (control) treatment had similar FS (18±1% and 19±1%, respectively, P>0.05) as compared to baseline value of 47±1%. At days 7 and 28 post-MI, sildenafil-treated group had a significantly higher FS than saline-treated mice (P<0.05). Both LVEDD and LVESD were increased in saline-treated mice as compared to sildenafil-treated mice (P<0.05), indicating more dilatation. Moreover, AWDT was greater in sildenafil-treated animals versus saline-treated animals (P<0.05) on day 28 post-MI. Fractional shortening of sham-operated mice was 43±1.0% at 28 days post left thoracotomy. An increase in LVEDD from a baseline value of 3.5±0.1 mm and a decrease in FS in salineand sildenafil-treated mice as compared to baseline and sham-operated mice (P<0.05) was observed on days 3 and 28, as shown in figure 5.

Fig. 5. Late sildenafil treatment preserves LV function and attenuates the progression of ischemic heart failure

#### **2.4 Sildenafil preserves LV function in infant rabbits**

Although we previously showed that PDE-5 inhibitors induce powerful preconditioninglike protective effects in the ischemic heart, it is not known whether sildenafil exerts similar

has a negligible role in a post-infarct setting (Anversa et al., 1993). To address this question, we administered sildenafil 3 days following MI (Chau et al., 2011). Specifically, we sought to determine if sildenafil treatment following LV dysfunction, defined as FS less than 25% at day 3 post-MI, could prevent the progression of HF in a permanent LAD occlusion model. At 3 days post MI, mice receiving sildenafil or saline (control) treatment had similar FS (18±1% and 19±1%, respectively, P>0.05) as compared to baseline value of 47±1%. At days 7 and 28 post-MI, sildenafil-treated group had a significantly higher FS than saline-treated mice (P<0.05). Both LVEDD and LVESD were increased in saline-treated mice as compared to sildenafil-treated mice (P<0.05), indicating more dilatation. Moreover, AWDT was greater in sildenafil-treated animals versus saline-treated animals (P<0.05) on day 28 post-MI. Fractional shortening of sham-operated mice was 43±1.0% at 28 days post left thoracotomy. An increase in LVEDD from a baseline value of 3.5±0.1 mm and a decrease in FS in salineand sildenafil-treated mice as compared to baseline and sham-operated mice (P<0.05) was

observed on days 3 and 28, as shown in figure 5.

**3 d MI 2 8d Sham 28 d MI +**

**\* P<0.05 vs. Saline at 7d # P<0.05 vs. Salin e at 28d ( n=10-12/group )**

**3 7 28 0.0**

**Days post MI**

**Sa line Silde na fil**

**\***

**3 4 7 28**

**Day s pos t MI**

**\* P< 0. 05 vs. Salin e a t 28d ( n =1 0-1 2/grou p )**

**0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9**

ischemic heart failure

**LV Ejection Fraction**

**( %)**

**AWDT ( mm)**

**A. B.**

**Sali ne**

**F. G.**

**# #**

**\***

**\***

**2.4 Sildenafil preserves LV function in infant rabbits**

**Fasu d il**

**Saline**

**Sildenafil Fasudil**

**C. D. E.**

**28 d MI + Sild enafil**

> **3.5 4.0 4.5 5.0 5.5 6.0**

**LV End Diast olic Diameter**

**( mm)** **28 d MI + Fasud il**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group )**

**3 4 7 28**

**3 4 7 28**

**Days post MI**

**3.0 3.5 4.0 4.5 5.0 5.5**

**LV End Systolic Diamet**

**(mm)**

 **er**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group)**

**\***

**Saline**

**# Sildenafil**

**# Fasudil**

**LV Fract ional Sho rtening**

**(%)**

**Days post MI**

**Saline Sildenafil (n = 10-12/group)**

**3 4 7 28**

**Days post MI \***

**Saline**

**Saline**

**Sildenafil**

**# # Fasudil**

**# Fasudil**

**\* P<0.05 vs. Saline at 7d**

**# P<0.05 vs. Saline at 28d (n=10-12/group)**

**Sildenafil**

**#**

**Fasudil**

**Heart Rate (bpm)**

Fig. 5. Late sildenafil treatment preserves LV function and attenuates the progression of

Although we previously showed that PDE-5 inhibitors induce powerful preconditioninglike protective effects in the ischemic heart, it is not known whether sildenafil exerts similar

**3 4 7 2 8**

**\***

protective effects against ischemia/reperfusion injury in the infant rabbit hearts as well. In this study, we used the model of coronary artery occlusion and reperfusion in infant rabbits (Bremer et al., 2005), which is similar to our previously described adult rabbit model of myocardial infarction. The benefits of this work are immense since this model may be applicable in pediatrics, and especially in pediatric cardiovascular surgery where there may be periods of ischemia/reperfusion injury. Also, we used two-dimensional (2D) and Doppler trans-esophageal echocardiography (TEE) for the estimation of LV cardiac output (LVCO) and aortic velocity time integral (VTI) in this model. A 10-Fr AcuNav diagnostic ultrasound probe (Acuson Corp., Siemens, Iselin, NJ) was inserted into the esophagus, at baseline, after the 30 min period of ischemia, and after 3 h of reperfusion in both the control and sildenafil groups. Standardized 2D imaging in a long axis view of the LV to show LV inflow across the mitral valve and LV outflow tract (LVOT) was obtained. Aortic flow Doppler across the aortic valve was performed in a long axis view of the LV and LVOT to obtain LVCO. The standard equation: mean velocity (cm/s) × flow area (cm2) × 60 (s/min), where mean velocity (cm/s) = VTI (in cm/beat) ÷ RR interval (s/beat), was used to obtain LVCO, expressed in milliliters per minute. Laminar Doppler flow across the aortic valve confirmed the absence of aortic stenosis. Color Doppler assessment was made of both the aortic and mitral valves again in the long axis view at baseline, after the ischemic period, and after 3 h of reperfusion for the presence or absence of mitral or aortic regurgitation. Subjective functional assessment was also made after ischemia and reperfusion to demonstrate at least left ventricular apical diminished contractility to confirm infarction. In this study, we showed that both the control and sildenafil-treated groups had comparable LVCO and aortic VTI at baseline. The controls had a decline in LVCO and aortic VTI immediately after the 30-min period of ischemia (28% and 27% lower than baseline values, respectively, p < 0.05), whereas the LVCO and aortic VTI increased in the sildenafil group after ischemia (43% and 45% higher than baseline values, respectively, n = 6 per group, p < 0.05). Both groups, however, had significant decline in LVCO after 3 h of reperfusion (54% of baseline in the sildenafil group, p < 0.05, and 62% of baseline in the control group, p < 0.05), and were not statistically significantly different from each other (n = 4–6 per group). Both groups demonstrated a decrease in aortic VTI after 3 h of reperfusion. However, this decline was only statistically significant in the control group compared with baseline values. None of the rabbits had aortic stenosis or developed aortic regurgitation for the duration of the study. Moreover, both the control and sildenafil groups demonstrated a comparable amount of mitral regurgitation (no more than mild) after ischemia/reperfusion, and none of the rabbits had baseline mitral regurgitation.

#### **2.5 Sildenafil and vardenafil preserve LV function in female mice**

Since the impact of PDE-5 inhibitors on the female cardiovascular system following ischemia remains unknown, we interrogated the effect of sildenafil and vardenafil on ischemia/reperfusion injury in female mice. In this study, adult female mice were pretreated (ip, bid) with sildenafil (0.7 mg/kg), vardenafil (0.14 mg/kg) or saline one hour before left coronary artery ligation for 30 minutes and reperfusion for 24 hours (Salloum et al., 2008b). Cardiac function, evaluated using echocardiography, showed that LV enddiastolic and end-systolic diameters increased 7 days post myocardial infarction with saline

Phosphodiesterase-5 Inhibitors Improve Left Ventricular Function in Failing Hearts 105

Fig. 6. Tadalafil attenuates doxorubicin-induced LV dysfunction

**4. PDE-5 inhibitors protect against hypertrophy-induced cardiac dysfunction**  Sustained pressure overload leads to cellular and molecular changes that are initially activated as compensatory mechanisms but later become maladaptive and contribute to progressive cardiac dysfunction and heart failure. This response involves a combination of complex signaling and transcription pathways that induce hypertrophic remodeling (Frey & Olson, 2003; Frey et al., 2004). The heart appears to have an intrinsic signaling system coupled to cGMP that can inhibit myocardial proliferative responses. Several studies using approaches that involve enhanced cGMP synthesis or prevention of its degradation have

(3.5±0.1 mm and 2.4±0.2 mm, respectively). In contrast, no dilatation was detected in sildenafil (3.0±0. mm and 1.4±0.1 mm, respectively) and vardenafil (2.9±0.3 mm and 1.4±0.2 mm, respectively) groups. Fractional shortening decreased at 7 days post infarction with saline (30±4%; P<0.05), but was preserved with sildenafil (52±2%) and vardenafil (53±5%). These data clearly suggest that PDE-5 inhibitors induce powerful cardioprotection in female mice as well. For this reason, PDE-5 inhibition may be a novel therapeutic strategy against ischemia/reperfusion injury in women with coronary artery disease.


**Author details**

Annabelle Mathieu-Denoncourt, Sean Giacomucci and Marylise Duperthuy\* Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Canada

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Tall fescue is a cool-season, perennial grass that is extensively utilized for grazing and hay production on approximately 15 million hectares in the eastern half of the USA. The grass is persistent and productive under low management, but this benefit is primarily due to alkaloids produced by a *Neotyphodium* endophyte (Hoveland et al., 1983; Strickland et al., 2011) that inhabits the intracellular spaces of most tall fescue plants. Groups of alkaloids (e.g., lolines and peramines) are recognized for providing the plant with tolerances to environmental stresses, such as drought and herbivory (Siegel et al., 1990; Bacon, 1993).

Ergot alkaloids are a class of alkaloids also produced by the endophyte that causes a toxicosis in grazing livestock that reduces both reproductive performance (Porter and Thompson, 1992) and growth rate (Schmidt and Osborn, 1993; Strickland et al., 1993; Paterson et al., 1995). Ergot alkaloids bind biogenic amine receptors in the vasculature to induce persistent vasoconstriction (Oliver, 2005). Constricted blood flow to peripheral tissues reduces the animal's ability to regulate body temperature and, therefore, be vulnerable to heat and cold stress (Aldrich et al., 1993). Ergovaline is an ergopeptine ergot alkaloid that was demonstrated to have the highest vasoconstrictive potency of the ergot alkaloids (Klotz et al., 2008) and also is of highest concentration, ranging from 84 to 97% of the ergopeptine fraction (Lyons et al., 1986).

Browning (2012) reported reduced growth rates of Boer, Kiko, and Spanish goats *(Capra aegagrus hircus)* fed diets containing 1.16 ppm ergovaline as compared to a diet free of ergot alkaloids. However, alterations in the physiology of goats grazing toxic endophyte-infected tall fescue have not been investigated (Ditsch and Aiken, 2009), but is needed as the goat industry expands into the transition zone and goats are exposed to toxic ergot alkaloids. Vasoconstrictive responses to ergot alkaloids have been reported in cattle (Rhodes et al., 1991; Aiken et al., 2009b), sheep (Aiken et al., 2011), and horses (McDowell et al., 2013), but not in goats. Therefore, a pen study was conducted using Color Doppler ultrasonography to compare luminal areas of carotid and auricular arteries in wether goats that were

1Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

fed either endophyte-infected (E+) or endophyte-free (E−) tall fescue seed.

#### **MATERIALS AND METHODS**

The experiment was conducted in indoor pens under a controlled ambient temperature of approximately 20◦C. All animal research followed procedures approved by the University of Kentucky Institutional Animal Care and Use Committee (protocol number 2013-1152).

Seven Spanish wether goats that were ruminally fistulated (age = yearling; body weight = 34.3 ± 1.7 kg) were randomly assigned to 4 goats in one pen group and 3 in the other group to compare luminal areas of the carotid and auricular arteries between diets containing either E+ or E− tall fescue seed. A 7 d adjustment period was conducted to measure DM intake of commercially chopped orchardgrass (*Dactylis glomerata* L.) hay. Two experimental periods followed the adjustment period as a crossover design, with the group of 3 goats receiving the E+ seed in the first period and with the treatments being switched between the two groups at the beginning of period 2. Rumens were infused daily at an average of 143 and 142 g of E− or E+ seed, respectively, during period 1 and 135 and 170 g of E− or E+ seed, respectively, during period 2. Duration of period 1 was 14 days and for period 2 was 13 days.

The cultivar "Defiance" was used for the E+ treatment and the cultivar "Kentucky 32" was used for the E− treatment. Ergovaline plus its epimer, ergovalanine were analyzed for both cultivars using procedures of Yates and Powell (1998) and modified as described by Carter et al. (2010).

Hay was fed *ad libitum* and seed that was previously course ground using a Wiley Mill with a 5.0 mm screen was infused into each rumen using a funnel. Feeding of hay and treatment with seed was done at 1500 h each day. During the experimental periods the previous day's intake for each pen were used to estimate intake per goat and the ratio of fed chopped hay to rumenally placed seed for providing a daily diet concentration of 0.8µg of ergovaline and ergovalanine, per gram of dry matter. Ergovaline plus ergovalanine concentrations in E+ and E− seed contained 2.71 and 0µg per gm DM, respectively, which resulted in seed averaging 14.5% of the diet dry matter for the E+ and E− treatments.

Color Doppler ultrasound images of the cross-sections of the right carotid (**Figure 1**) and auricular (**Figure 2**) arteries were collected using a Classic Medical TeraVet 3000 Ultrasound Unit (Classic Universal Ultrasound, Tequesta, FL) with a 12L5-VET (12 MHz) linear array transducer. Baseline measures were collected for each goat during the adjustment period on days 3, 5, and 7. Images were collected during period 1 on days 1, 2, 4, 6, 8, and 14, and were collected during period 2 on days 1, 2, 3, 4, 7, 8, and 10. Each imaging session was started at approximately 1100 h and was completed within 30 to 40 min. The goats were handled frequently prior to the start of the study to reduce excitability when being imaged. Individual goats were removed from their pens and their heads were stabilized by gently holding their horns without being placed in a chute. Each goat was clipped at the start of the experiment using surgical clippers under the left ear crosssectional images were collected for each artery using a frequency of 5.0 MHz and a pulse repetitive frequency that ranged between

**infusion of endophyte-infected or endophyte-free tall fescue seed.** Color delineates blood flow in the lumen of the artery.

2.5 and 3.0 kHz. Scan depth was set at 4 cm for the carotid arteries and 3 cm for the auricular artery. Following freezing of an individual scan, frames stored in the cine memory of the unit were searched to store the image exhibiting the maximum flow signal and assumed to be at peak systolic phase. The flow signal was traced to estimate lumen area (Aiken et al., 2009a).

Caudal and auricular artery luminal areas were analyzed using mixed models of SAS as repeated measures with the heterogeneous autoregressive covariance structure for responses of carotid arteries in periods 1 and 2 and auricular arteries in period 2 and with the autoregressive covariance structure for the auricular arteries in period 1 (Littell et al., 1996). The analysis used individual animals as the experimental unit. Treatment (E+ vs. E−), imaging day, and the interaction between treatment and imaging day were analyzed as fixed effects for both experiments. Measures for the adjustment period were averaged and used as the baseline measure (image day 0) for period 1 and those for period 1 were averaged for each treatment and used as the baseline measure for period 2. In the presence of a significant (*P <* 0*.*05) treatment x imaging day interaction, differences in least square means between baseline measures and imaging days and differences between treatments at each imaging day were determined using the PDIFF option of SAS.

#### **RESULTS**

Mean dry matter intake during the adjustment period was 0.97 kg/goat. During period 1, it was 0.80 and 0.81 kg/goat for E− and E+ treatments, respectively, and during period 2 it was 0.76 and 0.97 kg/goat, respectively. Consumption of ergovaline and ergovalanine averaged approximately 1*.*1 × 10−5µg per kg body weight.

#### **PERIOD 1**

Mean luminal areas of the carotid arteries in E+ goats were less (*P <* 0*.*05) than those in E− goats, and there was no treatment × imaging day interaction (*P* = 0.805; **Figure 3A**). Mean luminal area for goats on the E− treatment during the period was 9.4 ± 0.9 mm2 and for those on the E+ treatment it was 5.8 ± 1.1 mm2.

Different from carotid arteries, there was a treatment × imaging day interaction (*P <* 0*.*001) on luminal areas of the auricular arteries (**Figure 3B**). Luminal areas of auricular arteries for the E− treatment were not different (*P >* 0*.*12) from the mean baseline value, whereas those for the E+ treatment differed from mean baseline areas for all imaging days on and after day 2. Auricular arteries in E+ goats showed vasoconstriction (*P <* 0*.*05) as compared to E− goats on the imaging sessions for days 2, 4, and 12, even though after the second imaging day lumen areas in E+ goats were never greater than 2.3 mm2 and in E− goats they were never less than 2.5 mm2.

#### **PERIOD 2**

There was a treatment × imaging day interaction (*P <* 0*.*001) on carotid arteries (**Figure 4A**). Goats that were switched to E+ exposure after being on the E− treatment in period 1 showed less luminal areas than the baseline measure on and after imaging day 3. Luminal areas for the E+ treatment were less (*P <* 0*.*05) than those for the E− for imaging days 2, 3, 6, and 7, whereas there was a tendency (*P <* 0*.*10) for a difference on the day 9 imaging when the standard errors for both treatment groups were highest.

A treatment × imaging day interaction also was detected (*P <* 0*.*01) for luminal areas of auricular arteries (**Figure 4B**). Auricular arteries in goats switched to E− from the E+ treatment

in period 1 were relaxed from the baseline measure on and after day 2. Those in goats switched to E+ in period 2 had a tendency (*P <* 0*.*10) for vasoconstriction as compared to baseline measures on day 2, and exhibited significant (*P <* 0*.*05) vasoconstriction on and after day 3. Luminal areas in the E+ goats tended (*P <* 0*.*10) to be less (*P* ≤ 0*.*05) than for the E− goats day 1, but exhibited significant (*P <* 0*.*05) vasoconstriction on and after day 2 (*P <* 0*.*05).

#### **DISCUSSION**

Blood flow resistance under normal conditions is regulated by vasoconstriction and vasorelaxation/vasodialation of arteries and veins that is controlled by the autonomic nervous system. Blood flow volume is proportional to the fourth power of luminal radius, which results in large decreases in blood flow volume occurring with small decreases in luminal area (Carter, 2000). Therefore, responses of luminal areas of blood vessel crosssections provide a direct measure of vasoconstriction or vasorelaxation that has a direct bearing on blood flow volume.

Although ergovaline is the ergopeptine of highest concentration and has the highest potency, other ergot alkaloids

were assumed to be in the seed that could have additive effects on vasoconstriction (Klotz et al., 2010). Klotz et al. (2007) used an *in vitro* procedure with lateral saphenous veins biopsied from endophyte-naïve heifers to determine that ergovaline elicited a vasoconstrictive response at a concentration 1 × 10−8 M and had a maximum contractility of 69.9% relative to norepinephrine contractility. From an earlier experiment, Klotz et al. (2006) reported a concentration greater than 1 × 10−4 M was necessary for lysergic acid to induce vasoconstriction with a maximum contractility of 15.6% relative to norepinephrine contractility. Klotz et al. (2008) later reported that ergonovine, ergocryptine, ergocristine, and ergocornine induced a contractile response at similar concentrations (1 × 10−7 M) to ergovaline, but the greatest maximum contractility intensity relative to norepinephrine was achieved by ergonovine (68.5%), and maximum contractility intensities were similar between ergocryptine (45.5%), ergocristine (42.9%), and ergocornine (57.2%). Although ergovaline has demonstrated to be the most potent vasoconstrictor, Klotz et al. (2009) surmised that ergopeptines likely have additive effects on intensity of vascular contraction, thus indicating all are likely contributors to fescue toxicosis.

Overall, luminal areas tended to be more variable in E− goats than in E+ goats. This was clearly indicated by the responses of carotid arteries in both periods and the auricular arteries in period 2 which had Akaike's Information Criterion values in statistical analyses that favored the heterogeneous autoregressive covariance structure, which accounts for heterogeneity of treatment variances (Littell et al., 1996). Luminal areas of caudal arteries in cattle also have been observed to be more variable with E− than E+ diets (Aiken et al., 2007, 2009a). Alkaloid-induced vasoconstriction apparently reduces vascular responsiveness to environmental stimuli.

The goats used in the study were not endophyte-naïve. Prior to placement in the indoor pens they resided in a mixed grass pasture that had moderate percentages of endophyte-infected tall fescue; however, they had been on a non-toxic hay diet in the pens for 38 days prior to the 7 day adjustment period when their rumens were daily infused with E− seed. It was assumed that most of the ergot alkaloids had cleared from the vasculature prior to initiating the E+ diet, which was indicated by the alkaloidinduced vasoconstriction that occurred in both arteries during period 1. Overall luminal areas of carotid and auricular arteries were less (*P <* 0*.*05) than baseline measures taken during the adjustment periods by day 2.

Day-to-day fluctuations in luminal areas in both arteries in E+ goats that were parallel with those in E− goats are indicative of these goats not being saturated with ergot alkaloids and still having an ability to make vascular adjustments. Using an *in vitro* model with biopsied saphenous veins from endophyte-naïve heifers, Klotz et al. (2009) reported increased smooth muscle contractility with increasing repetitive additions of 1 × 10−7 M concentrations of ergovaline. It was further shown that ergovaline bioaccumulated in the saphenous veins with the repeated exposures and washings. Therefore, this indicates that ergovaline can bioaccumulate in the bovine vasculature through their affinity to biogenic amine receptors.

Switching treatments between the two goat groups in period 2 allowed for an evaluation of possible recovery of the vascular system to previous ergot alkaloid exposure. Detection of relaxation by the carotid and auricular arteries starting on image day 2 indicated that the goats were not saturated with ergot alkaloids by completion of feeding E+ seed during period 1. Aiken et al. (2011) reported luminal areas of auricular arteries in ewe lambs to linearly increase over time after they were switched to E− perennial ryegrass pasture after 19 days of grazing E+ pasture.

Vasoconstrictive responses in period 2, as detected by differences with baseline measures during period 1, were not as immediate as in period 1, with significant differences from baseline measures from period 1 not being observed until image day 3 for the carotid artery and day 6 for the auricular arteries. Treatment differences on individual imaging days cannot be interpreted because of confounding between relaxation of the arteries in goats that were switched from E+ to E− diets and vasoconstriction in those that were switched from E− to E+ diets. Nonetheless, the objectives of period 2 were to evaluate differences in luminal areas of the arteries between period 2 images and baselines means for detecting artery relaxation in E− goats and alkaloid-induced vasoconstriction in E+ goats.

#### **CONCLUSION**

Results of this experiment indicated the vasculature of goats can vasoconstrict when exposed to ergot alkaloids. Alkaloid-induced vasoconstriction of the carotid and auricular arteries was mediated within 1 to 2 days after being exposed to ergot alkaloids during period 1, and 2 to 3 days during period 2. Luminal areas of carotid and auricular artereries in E+ goats during period 1 showed rapid increases after they were switched to the E− in period 2, with significant relaxation being accomplished in 2 days for both arteries. The 12-day exposure to ergot alkaloids was short and likely was not long enough for vascular systems in the goats to be saturated with alkaloids. This experiment provides the first documentation of ergot alkaloid-induced vasoconstriction in goats.

#### **REFERENCES**

of fescue-naïve bovine lateral saphenous veins. *J. Anim. Sci*. 86, 1021–1028. doi: 10.2527/jas.2007-0576

**Conflict of Interest Statement:** The Reviewer Joan M. Burke declares that, despite being affiliated to the same institution as authors Glen E. Aiken and Michael D. Flythe, the review process was handled objectively and not conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 July 2014; accepted: 27 October 2014; published online: 20 November 2014.*

*Citation: Aiken GE and Flythe MD (2014) Vasoconstrictive responses by the carotid and auricular arteries in goats to ergot alkaloid exposure. Front. Chem. 2:101. doi: 10.3389/fchem.2014.00101*

*This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry.*

*Copyright © 2014 Aiken and Flythe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth

Charles F. Rosenkrans Jr.\* and Nicholas S. Ezell

*Department of Animal Science, University of Arkansas, Fayetteville, AR, USA*

Determining a grazing animal's susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40µM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of *in vitro* cytochrome P450 activity was affected (*P* < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (*n* = 39; 216 ± 2.6 days of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 days. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (*r* = −0.31; *P* < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (*P* < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

Keywords: fescue toxicosis, predictive biology, cattle

#### Introduction

Tall fescue [Lolium arundinaceum (Schreb.) S. J. Darbyshire] infected with the endophytic fungus (Neotyphodium coenophialum) is responsible for the production of ergot alkaloids which have a significant economic impact on beef cattle. Consumption of those mycotoxins is known to lower average daily gain, negatively impact reproductive traits, decrease parasite resistance, and reduce heat tolerance, all of which drive up the cost of production (for review see Strickland et al., 2011). Ergot alkaloids have an interesting relationship with animal cytochrome P450 (CYP) enzymes; CYP have been shown

#### Edited by:

*James Levi Klotz, United States Department of Agriculture – Agricultural Research Service, USA*

#### Reviewed by:

*Nick M. Filipov, University of Georgia, USA Raja S. Settivari, The Dow Chemical Company, USA*

#### \*Correspondence:

*Charles F. Rosenkrans Jr., Department of Animal Science, 1-University of Arkansas, B-107E AFLS Building, Fayetteville, AR 72701, USA<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry*

> Received: *08 July 2014* Accepted: *23 February 2015* Published: *11 March 2015*

#### Citation:

*Rosenkrans CF Jr. and Ezell NS (2015) Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth. Front. Chem. 3:16. doi: 10.3389/fchem.2015.00016*

February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 Volume 3 | Article 8 February 2015 Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 February 2015 | Volume 3 | Article 8 | | | | | | | || | | | | | |

to metabolize ergot alkaloids via hydroxylation, but CYP enzyme activity is inhibited by ergot alkaloids (Althaus et al., 2000; Settivari et al., 2006). Furthermore, CYP is a key enzyme in the biosynthetic pathway for ergot alkaloids (Haarmann et al., 2006).

Current methods for detecting ergot alkaloid concentrations in bodily fluids are limited. Initially, chromatographic methods had relatively high detection limits (Yates and Powell, 1988; Rottinghaus et al., 1991; Moubarak et al., 1996); however, more recent advances in chromatographic and detection methods have vastly improved the sensitivities of the assays (Lehner et al., 2005; Smith et al., 2009; Foote et al., 2012; Wang et al., 2013). Enzyme linked immunosorbent assay is a faster method for detection of ergot alkaloids and has been used to screen livestock exposure to fescue toxins (Shelby and Kelley, 1990; Hill and Agee, 1994). Recently, we demonstrated that the Promega™ P450-Glo assay could be used to assess ergot alkaloid content of tall fescue extracts (Moubarak et al., 2012). The Glo-assay is relatively easy with a quick protocol which makes it a convenient tool for biologically-relevant indirect alkaloid detection. Our objectives were to validate our previous findings related to ergot alkaloid inhibition of CYP450 activity, and determine if urine collected from steers grazing tall fescue inhibited CYP450 activity in vitro.

### Materials and Methods

### Experiment 1

#### CYP450 Analysis

Ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN) were dissolved in methanol (100%) resulting in final assay concentrations of 0, 20, and 40µM. Enzyme activity for CYP450 was assayed using the Promega™ P450-Glo assay (product # V9920; Cali et al., 2009). Briefly, sample (12.5µL) was added in triplicate to a 96-well plate, followed by CYP3A4 solution (12.5µL), and incubated at 37◦C for 10 min. Reaction was initiated by addition of NADPH regeneration system (25µL) and plate was incubated 30 min at 37◦C. Luciferin Detection Reagent (50µL) was added and plate was incubated at 20◦C for 20 min. Luminescence was recorded with a luminometer (Perkin-Elmer, Victor 1420 multilabel counter) using no filters and an integration time of 1 s/well.

#### Statistical Analysis

Luminescence data for each alkaloid were analyzed independently using mixed procedures. Main effects were replicate, alkaloid, and concentration; response variable was CYP450 activity as indicated by luminescence. If F-tests were significant (P < 0.05), multiple t-tests were used to separate means. Results are presented as a percent of CYP450 activity with zero alkaloid present.

#### Experiment 2 Steer Grazing

The University of Arkansas Animal Care and Use Committee approved all animal procedures (protocol # 04024). Healthy, fall-born Angus-sired crossbreed steers (n = 39; 203 ± 27 kg; 216 ± 2.6 days of age) were weaned, vaccinated, and treated with anthelmintic in May. Fourteen days after weaning, steers were randomly assigned to a pasture of either Kentucky 31 (K31; four pastures; n = 20 steers) or HiMag4 (HM4; four pastures; n = 19 steers) for a summer grazing trial. Pastures and tall fescue cultivars were as previously described (Nihsen et al., 2004). During the post-weaning grazing trial (105 days) steers received a daily supplement (1.8 kg/day; 80% ground corn: 20% soybean meal; 12% CP). During the grazing period steers had free access to water and minerals. Steers remained healthy throughout the trial and were not treated with pharmaceuticals.

#### Sample Collection

At weaning, approximately 10 mL of blood was collected from each steer, stored at 5◦C until centrifuged at 1200 × g for 30 min, serum was decanted and stored at −20◦C until hormone [cortisol, prolactin, and insulin-like growth factor 1 (IGF-1)] concentrations were determined by validated radioimmunoassay. At day 105, each animal was weighed and a urine sample collected. Urine samples were stored at -20◦C until assayed. Total alkaloids were determined by ELISA (Agrinostics Ltd. Co., Watkinsville, GA). Inhibition of CYP450 was determined using the assay (Promega™ P450-Glo) described above using 12.5µL of non-diluted urine in each reaction test.

#### Cytochrome P4503A28 Genotyping

Steers were genotyped at CYP3A28 single nucleotide polymorphism (SNP), C994G, using methods previously described (Sales et al., 2012). Briefly, genomic DNA was isolated from the buffy coat of EDTA treated whole blood samples (QIA-GEN Inc., Valencia, CA). Diluted DNA (20 ng/µL) served as the template for polymerase chain reaction with specific primers [forward (5′ -CAACAACATGAATCAGCCAGA-3 ′ ) and reverse (5′ -CCTACATTCCTGTGTGTGCAA-3′ )]. The 565-base pair amplicon was within the coding sequence of bovine CYP3A28 [National Center for Biotechnology Information (NCBI) gi1769423; (Natsuhori et al., 1997)]. Purified amplification products (QIAGEN Inc., Valencia, CA) were restriction enzyme digested with Alu I (New England Bio-Labs, Beverly, MA). Steer genotypes at SNP site C994G were homozygous cytosine, homozygous guanine, and heterozygous.

#### Statistical Analysis

Results of the CYP450 enzyme assay were reported in luminescence (arbitrary units). Pearson correlation coefficients were determined between dependent variables. Data were analyzed using mixed procedures with pasture as the experimental unit, genotype within pasture as random term, fescue cultivar as repeated, and creatinine as a covariate. If F-test for main or interactive effects were significant (P < 0.05) then means were separated using multiple t-tests.

#### Results

**Figure 1** validates our previous work (Moubarak et al., 2012) that ET, and DHET inhibit CYP450 activity as detected by the Promega™ P450-Glo assay. In contrast, CYP450 activity was numerically stimulated when 20 and 40µM of EN where added differ.

to the Glo assay. Our lowest test concentration, 20µM, resulted in a steep inhibition of CYP450 by ET and DHET suggesting that the sensitivity of assay was exceeded.

Steer weight gain (0.53 ± 0.14 kg/d) was typical for summer grazing of tall fescue with light grain supplementation. **Table 1** presents the Pearson correlation coefficients among body weight, gain, circulating concentrations of cortisol, prolactin, and IGF-1, urine inhibition of cytochrome P450 activity, and urine alkaloid concentrations. Cortisol concentrations at weaning were not correlated with body weight or gain. In contrast, prolactin concentrations and prolactin:cortisol were correlated (r > 0.30; P < 0.05) with body weight and ADG. Although steer body weights were correlated with IGF-1, ADG was not correlated to IGF-1. Inhibition of CYP450 activity by urine was correlated (r = −0.50; P < 0.05) with total ergot alkaloid concentration. Inhibition of CYP450 activity by urine also tended to be correlated (r = −0.26; P < 0.10) with ADG.

**Figure 2** displays the effects of C994G genotype on inhibition of CYP450 activity by urine. Steers that were heterozygous at C994G had more luminescence than steers that were either CC or GG. Luminescence was directly proportional to CYP450 activity; therefore, our work suggests that CG steers excreted urine that was less inhibitory to CYP450 activity. Tall fescue cultivar did not affect (P > 0.1) urine inhibition of CYP450.

#### Discussion

Determining the bioavailability of ergot alkaloids in the biological fluids of animals grazing tall fescue (E+) pastures infested with wild-type N. coenophialum is difficult. Previously, ergot alkaloids

*aSteers (n = 39) were weaned and grazed mixed tall fescue pastures for 105 days. Serum concentrations of cortisol, prolactin, and insulin-like growth factor 1 were determined at weaning. Urine was collected at day 105 of summer grazing, effects of urine on in vitro CYP450 expressed as percent of control (Urine inhibition), and total ergot alkaloids in urine (Urine alkaloids) expressed as ng of alkaloids per mg of creatinine.*

+*P* < *0.1.*

∗*P* < *0.05.*

∗∗*P* < *0.01.*

have been determined in cattle serum via HPLC (Moubarak et al., 1996), and in steer urine and bile (Stuedemann et al., 1998). Our results demonstrate that the Promega™ P450-Glo assay offers another method of indirectly determining ergot alkaloid concentrations in the urine of steers. Our preliminary research using the P450-Glo assay for the detection of animal to animal variation in circulating alkaloid concentrations proved too inconsistent to be useful (data not presented); however, refinement of the methodology may lead to a valid assay. Linearity of inhibition was problematic when testing cattle serum and plasma.

Stuedemann et al. (1998) successfully quantified total ergot alkaloid concentration in steer urine and bile using a validated ELISA. They found that 96% of all ergot alkaloids consumed were excreted through the urine. We analyzed urine from steers in our study using the same ELISA methods and found a strong negative correlation with luminescence from the Promega™ P450-Glo assay, suggesting the P450-Glo assay could indirectly determine animal clearance of ergot alkaloids. The ELISA method has been used primarily when scientists are interested in determining the concentration of total ergot alkaloids, not particular alkaloids (Stuedemann et al., 1998; Hill et al., 2000; Ayers et al., 2009). However, the ELISA assay lacks selectivity as it does not differentiate between ergot alkaloids which have necessitated the use of chromatographic methods to detect specific ergot alkaloids (Schultz et al., 2006; De Lorme et al., 2007; Foote et al., 2012; Wang et al., 2013). Schnitzius et al. (2001), compared HPLC and ELISA detection of ergot alkaloids and found the methods were inconsistent in their identification of alkaloids, concluding that both had their advantages and disadvantages. Antigen (ergot alkaloids) detection is restricted to the specificity of the antibody used in the ELISA test.

The P450-Glo assay also lacks selectivity as any compound that will inhibit CYP450 enzymes will be detected. However, the antibody used for ELISA is specific for the lysergic acid moiety (Hill and Agee, 1994), meaning any precursor molecules present, such as clavines, will not be detected using ELISA, but they would be detected in the P450-Glo assay if they inhibit CYP450. Based on our research, we cannot predict which alkaloid(s) will be associated with animal toxicosis. In fact, it is interesting that one of the more water-soluble ergot alkaloids, ergonovine, did not inhibit the P450-Glo assay yet factors in the urine did inhibit formation of luminescence. Perhaps other water-soluble alkaloids such as clavines and(or) lysergic acid and its derivatives are accumulating in urine, that would be consistent with previous metabolic research (Ayers et al., 2009). Ergopeptine alkaloids (e.g., ergovaline) are not excreted in the urine, so they would not be there to inhibit CYP450 (Schultz et al., 2006; De Lorme et al., 2007; Merrill et al., 2007); however, their metabolites may be in the urine following phase 1 and phase 2 toxin clearance in the liver.

Selecting livestock that are less susceptible to ergot alkaloid poisoning has the potential to improve livestock production where tall fescue is a component of the grazing system. In reality that is what livestock producers have been doing for the last 60 years. If the primary forage is tall fescue for a producer and the livestock do not reproduce then they are culled which results in selection for animals that tolerate the ergot alkaloids. Previously, we demonstrated that mutations in bovine cytochrome P4503A28 were associated with cattle profitability traits (Sales et al., 2012, 2013). In the current study, steer genotype at SNP C994G was associated with urine inhibition of CYP450 activity. Steers that were CG at C994G excreted urine that was less inhibitory to CYP450 activity. We speculate that heterozygous steers had metabolically altered the alkaloids which resulted in compounds that were not inhibitory of CYP450. Alternatively, CG steers may have deposited alkaloids in fat stores and were not excreted in the urine. Additional research will be required to determine ergot alkaloid deposition, and the dynamics and sensitivity of the P450-Glo-assay to biological fluids from cattle grazing various tall fescue cultivars.

The overall goal of our research is to develop management tools that can increase the sustainability of livestock production. In addition, we are interested in understanding the genetic and physiological mechanisms related to animal productivity and toxicology. Our results suggest that cytochrome P450 can be used as management tool for selecting animals based on their cytochrome P4503A28 genotype. In addition, we can envision the use of the P450-Glo-assay as a selection assay. We have not tested our hypothesis, but it is plausible that a group of replacement animals could be purposely exposed to a large concentration of ergot alkaloids and urine monitored for inhibition of the P450- Glo-assay. Presumably those animals with less inhibitory urine would be those with more tolerance of ergot alkaloids, but that would need to be tested before being recommended as a selection practice.

#### References

effects of high-ergot alkaloid tall fescue straw in beef cattle. J. Anim. Sci. 85, 2596–2605. doi: 10.2527/jas.2007-0075

lysergic acid in mature geldings. New Zealand Vet. J. 54, 178–184, doi: 10.1080/00480169.2006.36692

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Rosenkrans and Ezell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

### Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed

#### *A. Morrie Craig1 \*, James L. Klotz 2 and Jennifer M. Duringer 3*

*1 Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA*

*2 Forage-Animal Production Research Unit, United States Department of Agriculture-Agricultural Research Service, Lexington, KY, USA*

*3 Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, USA*

#### *Edited by:*

*Darrin Smith, Eastern Kentucky University, USA*

#### *Reviewed by:*

*Youngjoo Kwon, Ewha Womans University, South Korea Cynthia L. Gaskill, University of Kentucky, USA*

#### *\*Correspondence:*

*A. Morrie Craig, Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, 105 Magruder Hall, Corvallis, OR 97331,USA e-mail: a.morrie.craig@ oregonstate.edu*

Ergot-induced disease in humans was known long before Biblical times and has been the root cause for countless human epidemics spanning from the early fourteenth century to the late sixteenth century. In contrast, many of these same ergot alkaloids have been utilized for their medicinal properties to mitigate migraine headaches and have had indications as anti-carcinogens. Although ergot alkaloids have been used for centuries by humans, basic pharmacokinetic data has not been documented for clinical disease in livestock. Consequently, a threshold dose and accurate dose-response data have yet to be established. Throughout the past several years, new detection techniques have emerged to detect these alkaloids at the parts per billion (ppb) level which has allowed for new efforts to be made with respect to determining threshold levels and making accurate clinical diagnoses in affected animals. This perspectives article provides a critical initial step for establishing a uniform interpretation of ergot toxicosis from limited existing data.

**Keywords: ergot, ergotism, ergovaline, ergotamine, ergocornine, clinical disease, saphenous vein, cattle**

#### **INTRODUCTION**

Ergot alkaloids are made as secondary metabolites of fungi. Their production occurs in the sclerotia of several species of the genus *Claviceps*, the most common being *Claviceps purpurea*. These compounds belong to the family of indole alkaloids, one of the ten classes of alkaloids that pharmacologists have defined from plant secondary metabolites. Lysergic acid, a tetracycline ergoline ring system, is a common structure to all of the ergot alkaloids (Krska et al., 2008). The chemical structure of ergotamine, the most commonly found ergot alkaloid and the one responsible for the clinical disease known as ergot, has been documented in a classic text (Matossian, 1989; Bennett and Bentley, 1999; Council for Agricultural Science and Technology, 2003). Other major alkaloids detected in ergot sclerotia include ergocornine, ergocristine, ergosine, and ergonovine, and α-ergocryptine (Krska et al., 2008; Wallwey and Li, 2011; Takach and Young, 2014; also see **Figure 1**, the chromatogram).

Historically, ergot alkaloids have had a large impact on societies. They are likely the cause for the last of the 10 plagues of Egypt; it is believed that many of the oldest sons succumbed following the opening of grain and storage facilities whose contents were contaminated by the *Claviceps* fungi (Marr and Malloy, 1996). Accounts from the Middle Ages describe "St. Anthony's fire," a symptom of intense inflammation attributed to human consumptions of food prepared with ergot-contaminated rye. Furthermore, Marie Antoinette's comment, "Let them eat cake, not bread," most likely surfaced because of ergot contaminated rye bread which was a common food among peasants.

Currently, modern grain cleaning techniques have eliminated ergotism as a human disease, but it remains a significant and devastating disease in the veterinary field primarily affecting sheep, cattle, pigs, and chickens. Clinical signs of ergotism in animals include gangrenous extremities, abortion, convulsions, agalactia, and ataxia (Lorenz and Hoseney, 1979; Richard, 2007; Duringer et al., 2012). Agalactia and the loss of ears, tails, and hooves are common sequelae seen in animals due to vasoconstriction: ergot alkaloids affect the supply of blood to the extremities of the body in addition to acting directly on the central nervous system via the pituitary where they activate the D2 dopamine receptors (Oliver, 2005; Richard, 2012). The tetracyclic ergoline ring of ergot alkaloids is similar to the structure of biogenic amines (Eckert et al., 1978), allowing them to act on the dopaminergic (Larson et al., 1999), serotonergic (Dyer, 1993; Schöning et al., 2001), and α-adrenergic receptors (Oliver et al., 1998; Schöning et al., 2001). Most recently, plant agronomists have shown different varieties within a plant species can portray different "chemotypes" or constitutions different alkaloids (Wallwey and Li, 2011; Panaccione et al., 2012, 2014; Takach and Young, 2014). Sclerotia show significant differences in their total alkaloid content, varying between 0.01 and 0.05% (Krska and Crews, 2008). Patterns that occur in alkaloid production are attributed to an individual strain in a geographic region (Krska and Crews, 2008).

In the United States, there is no regulatory limit defined for ergot alkaloids in grain. In Europe, while there are discussions on establishing legal limits, the European Food Safety Agency (EFSA) at this time has not recommended a limit other than 0.03–3.6 and 0.6μg/kg b.w. per day for children and adults, respectively (European Food Safety Authority, 2012).

Two types of ergotism have been described: gangrenous (vasoconstrictive) and convulsive (neurological and abortogenic) (da Rocha et al., 2014). Though these two designations exist, the etiology of both is rooted in the vasoconstrictive ability of ergot alkaloids. Associating each alkaloid with a vasoconstrictive measurement (bovine lateral saphenous vein contractile response) has been a key factor in an accurate evaluation of clinical disease and will be discussed in **Table 1**.

The Endophyte Service Laboratory (ESL) (Craig et al., 1994, 2014), along with others (Rottinghaus et al., 1993; Saiga, 1998; Miyazaki, 2004), has conducted studies on threshold levels of alkaloids associated with tall fescue toxicosis. Moreover, these studies have been related to clinical signs as seen in fescue toxicosis (Brendemuehl et al., 1995; Blodgett, 2001; Tor-Agbidye et al., 2001; Marin et al., 2013). In contrast, few studies have been done on ergot-derived alkaloids to establish a threshold in livestock for ergotism. Thus, this article discusses the known correlations between the ergot alkaloids and development of ergotism in clinical cases to serve as a starting point for suggesting threshold levels and metabolism of these toxicants.

#### **METHODS**

#### **EXTRACTION OF THE ERGOT ALKALOIDS**

A method for extraction of ergot alkaloids from plant material was developed based on previous studies, for subsequent analysis by HPLC-fluorescence (Rottinghaus et al., 1991; Hill et al., 1993; Craig et al., 1994; Duringer et al., 2012). A standard mixture comprised of ergonovine, ergosine, ergotamine, ergocornine, αergocryptine, and ergocristine was purchased from Romer Labs, Inc. (Union, MO, USA). Seed, straw, hay, or feed pellet samples were ground in a Cyclotec 1093 sample mill and passed through a 0.5-mm screen. To each tube, one gram of sample, 11 mL of chloroform and 1 mL of 0.001 M NaOH were added and mixed for 18–24 h in the dark, then centrifuged at 650 × *g*. Five milliliters of supernatant was applied to a 500 mg/6 mL solid phase extraction (SPE) column containing 0.5 g Ergosil® and 0.5 g anhydrous sodium sulfate. The ergot alkaloids were extracted by adding 5 mL eluent to the SPE, followed by a 2 mL 4:1 acetone:chloroform (v/v) wash and elution with 2.5 mL methanol. The eluent was dried under nitrogen at 50◦C, then reconstituted in 0.5 mL methanol. Samples were mixed for 10 s,

**Table 1 | Concentration at onset of contractile response, half maximal effective concentration or potency (EC50), and the maximal response or efficacy (EMAX) of ergot alkaloids in bovine lateral saphenous veinsa.**

*aTaken from Klotz et al. (2006, 2007, 2010).*

*bExpressed as a percent of norepinephrine maximum, which was a* 1 × 10−4 *M norepinephrine reference addition. Onset is normalized to the closest order of magnitude.*

sonicated for 10 s, and centrifuged at 650 × *g* for 5 min then analyzed via HPLC-fluorescence.

#### **HPLC-FLUORESCENCE ANALYSIS FOR ERGOT ALKALOIDS**

Reverse-phase HPLC analysis is coupled with fluorescence detection (excitation and emission wavelengths of 250 nm and 420 nm, respectively) and a gradient run at 0.9 mL/min. Mobile phases of 1 mM ammonium carbonate (A) and ACN (B) were programmed as follows: equilibrate from 0 to 5 min at 75% A, then decrease linearly to 65% A from 5 to 15 min, hold at 65% A from 15 to 20 min, then decrease linearly to 25% A from 20 to 25 min. A Gemini 3.0 μ C18 110Å (Phenomenex, Torrance, CA) column was used in conjunction with a guard column cartridge of similar packing. **Figure 1** shows an example of a HPLC-fluorescence chromatogram for ergonovine, ergosine, ergotamine, ergocornine, α-ergocryptine, and ergocristine produced using this assay. [Ergonovine and the epimers of the other ergot alkaloids are not retained by the Ergosil® SPE columns (reference Krska et al., 2008) and are not included in reporting the total ergot alkaloid content of the feed sample]. The method performance has a limit of detection (LOD) of 11 ppb for ergosine, ergotamine, and ergocornine, 13 ppb for α-ergocryptine, and 14 ppb for ergocristine. The limit of quantitation (LOQ) is 39 ppb for ergosine and ergotamine, 41 ppb for ergocornine, 49 ppb α-ergocryptine, and 50 ppb for ergocristine. Within day/Day to day variations are 8.5%/6.5% for ergosine and ergotamine, 4.5%/6.9% for ergocornine, 5.8%/7.8% for α-ergocryptine, and 6.8%/10.6% for ergocristine. The recovery for the above-mentioned alkaloids is 92–97%.

#### **WHAT WE KNOW ABOUT VASOCONSTRICTION AND ABORTION FROM ERGOT ALKALOIDS**

Using the data presented in Klotz et al. (2006, 2007, 2010, 2011), we have constructed **Table 1**, which summarizes the vascular potency and efficacy of ergovaline (the predominant ergot alkaloid found in tall fescue), ergotamine, ergonovine, ergocristine, ergocornine, ergocryptine, and lysergic acid in relative terms. **Table 1** bridges what is known in tall fescue toxicosis, i.e., vasoconstriction and the extrapolated abortogenic sequelae. It should be noted that the principal column evaluated in determining the onset of vasoconstriction in clinical disease is column two [Onset (M)].

As **Table 1** emphasizes, ergovaline and ergotamine have equal sensitivity for vasoconstriction, whereas ergonovine, ergocristine, and ergocornine are about one-tenth as powerful. Pharmacologically, the half-maximal concentration (EC50) and the onset concentration are measures of a compound's potency (the lower the concentration the more potent the compound); ergotamine and ergovaline are identical in this aspect. The onset of vasoconstriction is reported in concentration (moles/L), and is viewed as the best indicator of toxicosis when evaluating levels in feedstuffs. Interpreting the chromatogram in **Figure 1** in relation to the additive effects of all ergot alkaloids present is performed by utilizing a multiplicative factor in front of various alkaloids; therefore, 10 ergonovine molecules are equally vasoconstrictive to one ergovaline molecule. More recently, Klotz et al. (2013) has demonstrated that the EC50 for ergovaline, ergotamine, and ergocornine can be reduced by an animal's prior exposure to ergot alkaloids. This may have an influence on the animal's perceived sensitivity (as reported by the clinician/researcher) and may affect the threshold of an individual animal (Pesqueira et al., 2014). Further, severe winters and wet springs (wet and cold weather patterns in general) have been shown to be more conducive to ergot-related disease (Welty et al., 1994). This strong correlation between weather and ergot disease is easily seen throughout historical events and in our own clinical cases below (**Table 2**).

#### **CORRELATION OF ERGOT TOXINS WITH CLINICAL DISEASE**

The alkaloids that cause fescue foot and summer slump syndrome are produced by fungi in pasture grasses. Because of similar vasoconstrictive abilities of ergovaline and ergotamine, we have correlated some of the studies done with fescue with what we have seen from clinical cases with ergot. Field and barn studies of natural fescue foot and herds have been conducted (Oliver, 2005; Marin et al., 2013). Therefore, we are illustrating typical cases seen in feed sample submissions over the last 2–3 years to the ESL in Oregon.

In **Table 2**, column 2 represents the total alkaloids observed, which include a summation of all six ergot alkaloids. Since different alkaloids have different vasoconstrictive onset values, column 3 relates the six alkaloids (**Table 1**) back to "ergotamine

*aIncludes ergonovine, ergosine, ergotamine, ergocornine,* α*-ergocryptine, and ergocristine.*

equivalents." Therefore, ergotamine equivalents are correlated to clinical signs (column 5).

Eight examples of clinical cases associated with feed samples submitted to the ESL by veterinarians suspecting ergot toxicosis are shown to illustrate typical cases that have been seen. The ESL consults with veterinarians and evaluates all aspects of the case to determine if testing of feed samples is warranted. If so, results are often discussed with the client to ensure that proper measures are taken to resolve ergot toxicosis. Five to ten years ago the ESL would observe ergot cases approximately once per month; however, this has progressed to several cases per week and sometimes multiple cases each day in recent years despite maintaining approximately the same number of total samples tested. Our worst toxicosis cases have been in cattle and horses. The increase in cases seen in the Pacific Northwest and throughout the country is likely due to climate change or the lack of field burning that has been phased out throughout the years. The stoppage of burning has greatly reduced the ability to destroy *C. purpurea* by fire.

Evaluation of the cases included in **Table 2** is as follows:

The following cases in cattle involved pellets as nearly 100% of the provided feed source, with the exclusion of case b, which used a grass seed sample. Pellets were usually a combination of grass seed with additional alfalfa or other hay sources mixed in. In cases f and h, cattle had exposure to overgrazed pasture, and it is estimated that little nutrition came from the pasture sources. In both cases, the pellet mixture comprised most of the usable diet.

The first case (a) presents an ergotamine level of nearly 500 parts per billion (ppb). This case originated in February when the colder weather prompted vasoconstriction in the extremities, specifically, the tissue around the tail to become necrotic. No sloughing of ears were observed nor reported in this particular case.

The second case (b) reported 1500 ppb ergot alkaloids and occurred in December as the feed bunks were being cleaned out. There was moderate lameness and some necrosis about the feet. The 1500 ppb consisted of ergotamine and ergocornine, resulting in a lower ergotamine equivalence level (415 ppb) that was similar to case (a).

The third case (c) revealed approximately 2900 ppb ergot alkaloids in pellets which triggered a decreased consumption in feed, as well as a necrotic area around the coronet band of the hoof. This outcome was observed in steers in January. Ergotamine, ergocornine, and ergocryptine were found in the feed. The ergotamine equivalence level (466 ppb) illustrated similarities to that of case (a).

The fourth case (d) presented nearly 3500 ppb ergot alkaloids and took place in February. This case showed greater severity of clinical signs, i.e., term abortions, agalactia, and reduced milk production in the dairy herd. This case had a 778 ppb ergotamine equivalence level.

The fifth case (e) presented itself in January and had a total ergot alkaloid concentration of 6000 ppb. The animals refused to consume any feed, resulting in near starvation. This case was composed of very little ergotamine and mostly other ergot alkaloids, which dramatically brought the ergotamine equivalence level down to 626 ppb.

The sixth case (f) occurred during April, with ergot alkaloid levels measuring almost 12,000 ppb. The steers exhibited sloughing of the hooves, tails, and ears. So many animals sloughed their hooves that approximately 40% of the herd had to be terminated. This case consisted of multiple ergot alkaloids and had an ergotamine equivalence level of approximately 1000 ppb.

The seventh case (g) was the second highest case of total ergot alkaloids (54,916 ppb) that we have observed and occurred in January. Though the weather was fairly warm and dry, there were early term abortions in a high percentage of the cow-calf operation. Approximately 45–47 of the 59 animals that were pregnant had early term abortions over a 2 week period. This case had an approximate 4000 ppb ergotamine equivalence level.

The eighth (h) and final case entailed the observance of cattle during January in the northern region of the United States. This case produced approximately 62,000 ppb of total ergot alkaloids. The clinical consequences of the ergot alkaloids resulted in 3/4 of the cow's hooves to slough off, after which the animals had to be sacrificed. This case consisted of both ergotamine and other ergot alkaloids, and had an ergotamine equivalence level to approximately 10,000 ppb. Although the weather was warm, the high concentration of ergot alkaloid induced enough vasoconstriction to clinically affect the animals.

The bioavailability and pharmacokinetic data of ergot alkaloids in cattle are limited and affected by many different factors. However, using the highest and the lowest dietary ergotamine concentrations reported in **Table 2**, calculations were done to illustrate a theoretical relationship with the ergotamine concentration in a feedstuff and a maximum potential concentration in blood. A feedstuff containing the lowest concentration of 473 ppb (case a) or 0.473 mg/kg ergotamine fed to a 500 kg cow consuming 2.5% of BW will consume a total of 5.9 mg of ergotamine per day. Assuming that the ergotamine is 100% bioavailable from the feedstuff (not always the case as this is negatively correlated with the maturity of the feedstuff), 100% absorbed (not always the case, as these compounds are likely biotransformed by gut microbes prior to absorption), 100% bioavailable in the blood (assuming negligible bioaccumulation in tissues or initial hepatic detoxification) and total blood volume of 55 mL/kg of BW, a maximum ergotamine concentration ever reaching the blood would be 0.215μg ergotamine/mL of blood. Although this is far below the EC50 reported for ergotamine in **Table 1** of 4 × 10−6 M or an approximate of 2.32μg of ergotamine/mL in blood, it is much higher than the corresponding onset concentration for ergotamine of 1 × 10−8 M or 0.005μg /mL. If we substituted the highest reported ergotamine concentration in **Table 2** (case h), the maximum concentration of ergotamine that could ever reach the blood is 4.6μg /mL. This is twice the concentration of the EC50 of ergotamine and could easily explain the severe clinical signs observed.

The data that resulted from these clinical cases illustrate that ergot alkaloids can cause vasoconstriction and deleterious consequences regardless of seasonal conditions (Egert et al., 2014). Severe vasoconstriction will cause the hooves to slough off and/or abortions. However, in cooler or freezing weather, the values for the vasoconstrictiveness of the different ergot alkaloids are not dissimilar from what is found in endophyte-infected tall fescue with the predominant ergot alkaloid ergovaline (Duringer et al., 2012; Foote et al., 2012). This observation should be documented in future scientific studies. As for now, there have been no specific studies looking at individual alkaloids other than the physiometry values documenting vasoconstriction. Furthermore, there have been no studies related to the convulsive aspect of this disease (neurological and abortogenic) and the individual alkaloids. The only clinical evaluation conducted has been by the papers from Klotz et al. (2006, 2007, 2008, 2010). These papers examine the onset of vasoconstriction. Threshold levels have yet to be established for different animal species. When considering the international transportation of feed, ergot levels become an important factor to evaluate in developing Food Safety standards and programs. In particular, feed produced in the European continent and transported to the Middle East are susceptible to *C. purpurea* due to the wet springs and summers that promote fungal growth.

#### **WHAT SHOULD BE DONE IN FUTURE STUDIES?**

Though this disease has been known for hundreds of years, only a paltry amount of information is available to accurately diagnose disease at a clinical level (where animal production is affected and animal welfare becomes a concern). The first step in expanding this information should be establishing the threshold level in different species, particularly cattle and horses that could be exposed to ergot alkaloids in their feed. It should be noted that overseas, camels, goats, and sheep are also frequently exposed to these toxic alkaloids. The second step is to establish other bio-indicators of clinical disease other than prolactin levels, weight gain, and some of the more classical animal observances. Lastly, the vasoconstrictive and convulsive effects and their subsequent clinical consequences need to be separated.

#### **ACKNOWLEDGMENT**

Special thanks and recognition go out to Avalon McKune and McKenzie Hughes for helping in the progress of this article.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 18 September 2014; accepted: 22 January 2015; published online: 18 February 2015.*

*Citation: Craig AM, Klotz JL and Duringer JM (2015) Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed. Front. Chem. 3:8. doi: 10.3389/ fchem.2015.00008*

*This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry.*

*Copyright © 2015 Craig, Klotz and Duringer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### ADVANTAGES OF PUBLISHING IN FRONTIERS

FAST PUBLICATION Average 90 days from submission to publication

COLLABORATIVE PEER-REVIEW

Designed to be rigorous – yet also collaborative, fair and constructive

RESEARCH NETWORK Our network increases readership for your article

#### OPEN ACCESS

Articles are free to read, for greatest visibility

#### TRANSPARENT

Editors and reviewers acknowledged by name on published articles

GLOBAL SPREAD Six million monthly page views worldwide

#### COPYRIGHT TO AUTHORS

No limit to article distribution and re-use

IMPACT METRICS Advanced metrics track your article's impact

SUPPORT By our Swiss-based editorial team

EPFL Innovation Park · Building I · 1015 Lausanne · Switzerland T +41 21 510 17 00 ·<EMAIL_ADDRESS>· frontiersin.org


*2.6. qRT-PCR Analysis 2.6. qRT-PCR Analysis* In order to validate the RNA-seq data and confirm the differential expression genes, we

**3. Discussion**

In order to validate the RNA-seq data and confirm the differential expression genes, we performed qRT-PCR on sixteen candidate DEGs associated with salt stress. The results revealed that these DEGs include AHL, dehydrin-1, highly ABA-induced PP2C gene, proline transporter 2-like, sodium/hydrogen exchanger 4, serine/threonine-protein kinase, heat shock protein, Cation/H(+) antiporter, wall-associated receptor kinase-like and dehydrin-3 were up-regulated in the leaves with salt treatment (Figure 6), whereas major allergen variant Cor, dehydrin-2, phosphoglycerate kinase, potassium transporter, peroxidase 4 and WRKY transcription factor were down- regulated in the leaves with salt treatment. The results indicated that these sixteen candidate DEGs had the same expression patterns compared with the sequencing data, suggesting the reliability of the RNA-seq data. performed qRT-PCR on sixteen candidate DEGs associated with salt stress. The results revealed that these DEGs include AHL, dehydrin-1, highly ABA-induced PP2C gene, proline transporter 2-like, sodium/hydrogen exchanger 4, serine/threonine-protein kinase, heat shock protein, Cation/H(+) antiporter, wall-associated receptor kinase-like and dehydrin-3 were up-regulated in the leaves with salt treatment (Figure 6), whereas major allergen variant Cor, dehydrin-2, phosphoglycerate kinase, potassium transporter, peroxidase 4 and WRKY transcription factor were down- regulated in the leaves with salt treatment. The results indicated that these sixteen candidate DEGs had the same expression patterns compared with the sequencing data, suggesting the reliability of the RNA-seq data.

**Figure 6.** qRT-PCR validation of sixteen selected DEGs in leaves. Fold changes of the DEGs are shown. The expression levels in CK were arbitrarily set to 1. Error bars represent the standard deviations of three technical PCR replicates. **Figure 6.** qRT-PCR validation of sixteen selected DEGs in leaves. Fold changes of the DEGs are shown. The expression levels in CK were arbitrarily set to 1. Error bars represent the standard deviations of three technical PCR replicates.