*2.3.1 Therapeutic opportunities based on Bcl-2 family proteins modulation*

In view of the critical role of Bcl-2 proteins in regulation of mitochondrial pathway of apoptosis, targeting various members of this family have been considered amongst the most promising therapeutic strategies in cancer, a well-known dysfunctional apoptosis disorder [117]. Numerous attempts have been carried out to target the modifications in Bim expression and therefore regulate tumor cell response to apoptosis. Histone deacetylase inhibitors have been shown not only cause to up regulation of Bim in transformed cells, but also they are able to reverse silencing of Bim in cancer cells and consequently restored their sensitivity to various anticancer-agents reported in leukemia and Burkitt's lymphoma cells [118]. The proteasome inhibitors are also recognized to promote accumulation of Bim and enhance the lethality of cancer cells [119, 120]. Another approach is through diminishing its degradation by blocking its phosphorylation. Ras/Raf/MEK/ERK pathway have a key role in regulating the expression and function of Bim through its phosphorylation and triggering its proteasomal degradation. MEK1/2 Inhibitors has been applied to disrupt this process leading to accumulation of Bim and consequently apoptosis. MEK1/2 Inhibitors are also able to modify the interaction between BIM and other Bcl-2 family members contributing to cell death [118, 121, 122].

Furthermore, structure-based drug design can be applied to discover anticancer agents which are able to effectively activate a pro-apoptotic Bcl-2 protein through changing its conformation promoting cell death. Bax as a unique entry point for intrinsic apoptotic signaling is another major pro-apoptotic member of the Bcl-2 family proteins which has been greatly getting attention to be targeted in order to control apoptosis. Recent studies have revealed that direct binding and activation of Bax can be a promising approach for cancer treatment. Discovery of small-molecule functioning as a Bax activators may result in selective induction of tumor cell apoptosis and overcome chemoresistance which has been proved through invitro and invivo studies [117, 123]. Besides, some studies targeting a regulatory site in Ser184 of Bax protein have determined that its agonists SMBA1–SMBA3 can effectively bind to and trigger its oligomerization through the suppression of its phosphorylation that eventually lead to cyrochrome c release and induction of apoptosis in mouse lung cancer xenografts [124]. Similar results were also reported with other Bax agonists as promising Bax direct activators in breast cancer, glioblastoma and acute myeloid leukemia cells. These drug candidates demonstrated noteworthy in vivo efficiency inhibiting xenograft tumor growth though induction of apoptotic cell death [125–127].

The next emerging strategy in cancer drug discovery was the BH3 mimetics which are able to antagonize the function of Bcl-2 and selectively kill cancer cells. In this approach, BH3 mimetics are antagonists of the anti-apoptotic Bcl-2 proteins. These small molecules acting as the competitive inhibitors induce apoptosis though binding to their hydrophobic cleft and therefore affect the interactions between

anti- and pro-apoptotic proteins [128]. Various BH3 mimetics with different level of specificity and efficiency have been reported. For instance, TW-37 derived from phenolic aldehyde gossypol has been showing high affinity to bind MCL-1, Bcl-2 and Bcl-xL anti-apoptotic proteins and induce apoptosis in B-cell lymphomas and pancreatic cell lines along with decreasing tumor size in xenograft models [129–131]. As ABT-737 mimicked the BH3 domain of Bad protein, it was able to bind selectively to Bcl-2, Bcl-xL and Bcl-W. It also demonstrated poor affinity to other member of ani-apoptic proteins including MCL-1 and BFL-1. ABT-737 has shown efficacy in the killing of several cancer cell lines including leukemia, lymphoma, multiple myeloma, glioma and small cell lung cancer cell lines as well as primary samples. Also, these two inhibitors of Bcl-2 families are currently in clinical trials [132–134].

Another approach to antagonize the function of Bcl-2 anti-apoptotic proteins is focusing on the protein interaction among members of Bcl-2 family through their essential death domain. In this regard, peptide-based inhibitors have been significant achievements in targeting and regulating intracellular protein–protein interaction. Stapled peptides are synthetic, bioactive α-helical peptides locked into their bioactive structure that have brought new hope to target drug discovery [135, 136]. For instance, stabilized alpha-helix of Bcl-2 domains, SAHBs, is the peptide having the ability to penetrate leukaemia cells and trigger induction of apoptosis through its binding to the Bcl-xL which its function has been further confirmed though invivo mouse xenograft models of leukaemia [137]. Another research study has also revealed that exclusive MCL-1 stapled peptide inhibitor (MCL-1 SAHBD) can effectively resensitize cancer cells to caspase-mediated apoptosis through directly targeting of MCL-1 and suppress its inhibitory interaction with Bak protein [138].

### *2.3.2 Therapeutic opportunities based on caspase modulation*

Given the vital role of caspases in the regulation of apoptosis, it is not surprising that numerous therapeutic opportunities targeting caspase activity demonstrate great promise for the cancer treatment. Different strategies have been investigated to upregulate caspase-8 expression to restore its function in tumors. As hypermethylation of its promotor has been recognized as the main mechanism of silencing, one approach for its reactivation is using demethylation agents. Azacytidine, decitabine and nucleoside analogs promoting the demethylation of caspase-8 promotor have been successfully applied in neuroblastoma, medulloblastoma, breast cancer and lung carcinoma [139, 140]. Another interesting strategy is designing the small molecules that selectively and directly target and trigger caspase-8 activation. These small molecules has been reported to potentiate TRAIL-induced cell death [141]. Proteosomal inhibitors such as bortezomib has been also reported to increase total cellular caspase-8 levels apparently by blocking its degradation [111, 142]. Some studies have also reported that the use of interferons can elevate the caspase-8 expression through modification at transcriptional level. This strategy targeting interferon-sensitive response elements within the caspase-8 promoter leading to sensitize cancer cell to apoptotic cell death in cancer chemotherapy or irradiation therapy [139, 143, 144].

Besides, developing molecules that are able to directly activates caspase 3 have been of research interests as well. For this purpose, particular sequence of inactive procaspase-3 consisting of the triplet of aspartic acid residues has been targeted. In vitro studies have exhibited that PETCM, gambonic acid and its derivatives have the potential to effectively activate caspase 3 leading to apoptotic cell death in cancer cell lines [145–147]. Furthermore, procaspase-activating compound1 (PAC-1) has been shown to induce anticancer activity through promoting the procaspase-3 activation. PAC-1 exerted its effect by chelation of inhibitory labile zinc ions and currently is in phase I clinical trial for cancer treatment [148].

#### *The Role of Apoptosis as a Double-Edge Sword in Cancer DOI: http://dx.doi.org/10.5772/intechopen.97844*

In order to sensitize tumor cells to apoptotic stimuli, caspase −9 can be also regarded as a potential target in cancer therapy. There are a wide range of molecules such as protein kinase, microRNAs and heat shock protein that have been identified to modulate caspase-9 expression and hence have been getting interest as candidates for new drug development though regulating intrinsic apoptosis in cancer cells [149, 150]. Targeting caspase-9 have been also initiated in clinical trials (phase I) against several cancer including Chronic Myeloid Leukemia, non-Hodgkin's lymphoma, Acute Lymphoblastic Leukemia, [151, 152].

In addition, several attempts have also been conducted on cancer gene therapy focusing on apoptotic caspases. Gene transfer technologies may restore caspase gene expression resulting in selectively induction of apoptosis in various tumor types [153–155]. In this regard, caspase-9 and caspase-3 has been suggested for being used in gene therapy strategies. A main benefit of involving these caspases is that they start apoptosis at the downstream of the mitochondrial outer membrane potential and they will not be affected with the enhanced expression of anti-apoptotic of Bcl-2 proteins. The researchers conducted on inducible version of these caspases have shown encouraging results related to remarkable reduction in size of lung and gastric tumors, respectively [156–158].

Other than directly targeting of caspases, another area of research has focused on discovery of anticancer agents that trigger the caspases activity indirectly. In this approach, certain members of the inhibitors of apoptosis proteins (IAP) are targeted. IAPs are functioning as the endogenous caspase inhibitors and prevent apoptosis event by binding and inhibiting caspases through the degradation of active caspases or keeping them away from their substrate. In this regard, numerous researches have investigated various IAP inhibiting agents, accomplishing a breakthrough in cancer treatment [159, 160]. Some of these agents are acting as the IAP antagonist and exert their effect via suppression of their activity, while others are analogs of the endogenous IAP inhibitor Smac. Several Smac mimetics such as LCL161 and birinapant IAP inhibitors have currently being tested in phaseI/II in clinical trials, with promising outcomes [161–164]. Besides, IAP inhibitors have been reported to exert the synergistic effect in combination chemotherapy and sensitize the cancer cells to radiotherapy which is of particular interest in malignant gliomas [165–167].

## **3. Conclusion**

It is well established that the apoptosis dysfunction promotes the malignant transformation and renders the cancer cell resistant to treatment. Targeting apoptotic pathways in tumor cells has been a main clinical interest as the evasion of apoptosis is a hallmark of all cancers regardless of their causes or types. There are numerous defects found in apoptotic mechanism contributing to inhibition of cancer cell death. As demonstrated in this chapter, impaired activation of caspases and disturbance in the balance between anti-apoptotic and pro-apoptotic members of Bcl-2 family proteins are remarkably involved in tumorgenesis. The enhanced knowledge about their critical roles in apoptosis and cell fate in recent years has eventually made them promising therapeutic targets. This also has facilitated the generation of more specific anticancer agents and led to shifting in anticancer therapy form typical cytotoxic approaches to the designing and development of apoptosis-inducing drugs that particularly target the cancer cells. An exciting development in successful eradication of cancer cells involves structure-based drug design of small molecules such as BH3 mimetics, specifically targeting Bcl-2 proteins, that is currently being tested in clinical trials with promising effects of

selective induction of tumor cell apoptosis and overcoming chemoresistance as well. These inhibitor molecules are in continuous development and a great deal of effort is required to discover the most efficient ones having more specificity for individual Bcl-2 proteins and offer maximal clinical efficacy. Besides, new therapeutic applications targeting apoptotic caspases including gene therapy approaches and small molecules suppressing inhibitors of caspases are beginning to show some promise through selectively and directly targeting of individual caspases and eventually triggering their activity. Caspase-targeted approaches, epigenetic modulators and their combinations with established therapies may have the potential to overcome the limitation of previous strategies through exerting synergistic pro-apoptotic activity and may enhance the effectiveness of conventional cancer therapy, worthy of further investigation in preclinical advanced models and clinical trial. Apoptosis-targeted therapies are now remarkably advancing and remain a promising approaches in future clinical practice of oncology.


**1.2.2 New parameters for the diagnosis of anemia**

The question regarding anemia therapy in those patients is which are the best parameters to assess the iron available for erythropoiesis. New laboratory parameters are reported by different manufacturers as potential tools for anemia and iron restricted erythropoiesis diagnosis. These tests include reticulocyte hemoglobin content, percentage of hypochromic red cells and soluble transferrin receptor (Wish, 2006; Goodnough *et al*., 2010).

Serum transferrin receptor (sTfR) is a useful test for this purpose because it is not affected by inflammation so is a reliable marker of iron deficiency in mixed situations (Punnonen *et al*., 1997; Beguin, 2003; Skikne, 2008).

The sTfR test is based on the fact that erythroblasts in the bone marrow will increase the presentation of membrane transferrin receptor in the setting of iron deficiency. If a patient is not receiving sufficient iron and erythropoiesis is being stimulated by an ESA, then increased transferrin receptors will become expressed on the erythroblasts, some of which come off and will be detectable in the circulation. The sTfR correlates with this membrane expression of the

Assessing Iron Status in CKD Patients: New Laboratory Parameters 233

This is not the case for MHC. MCH is calculated from red blood cell count and hemoglobin and represents the average; the percentage subsets of erythrocytes can give complementary information of the contribution of cell with extreme values (hypochromic and hyperchromic cells) to the mean values, reflecting the fluctuations of iron availability to the erythron in the

Modern counters provide information about the reticulocyte counts but also about the characteristics of these cells (size or hemoglobin content) related to the quality of the

Nevertheless, each Company applies the technology in a different way in the analyzers, with different algorithms to translate the electronic signals to graphs and numerical values. For this reason these new parameters are exclusive of each manufacturer and they are patented.

On last decades, several new red blood cell and reticulocyte parameters have been reported having utilities in detection of iron deficiency and functional iron deficiency. Two of these parameters are hypochromic red cells (referred to as %Hypo) and CHr (reticulocyte hemoglobin content) reported by the Siemens ADVIA 120 hematology analyzer (Thomas &

Reticulocyte hemoglobin content (CHr) and the percentage of hypochromic red blood cells (%Hypo) reflect iron availability and are reliable markers of functional iron deficiency

CHr is defined by the formula (CHr = MCVr X CHCMr), wherein MCVr is the mean reticulocyte cell volume and CHCMr is the mean hemoglobin concentration of reticulocytes,

Reticulocytes are immature red blood cells with a life span of only 1 to 2 days. When these are first released from the bone marrow, measurement of their hemoglobin content can provide the amount of iron immediately available for erythropoiesis. A less than normal hemoglobin content in these reticulocytes is an indication of inadequate iron supply relative to demand. The amount of hemoglobin in these reticulocytes also corresponds to the amount of hemoglobin in mature red blood cells. CHr has been evaluated recently in numerous studies as a test for iron deficiency and functional iron deficiency and has been found to be highly sensitive and specific. However, exact threshold values have not been established, as the

The measurement of CHr is a direct assessment of the incorporation of iron into erythrocyte hemoglobin and thus an estimate of the recent functional availability of iron into the erythron; due to the life span of the reticulocytes CHr is a sensitive indicator of iron

Epoetin is effective in stimulating production, of red blood cells, but without an adequate iron supply to bind to heme, the red blood cells will be hypochromic, i.e., low in hemoglobin content. Thus, in states of iron deficiency, a significant percentage of red blood cells leaving the bone marrow will have a low hemoglobin content. By measuring the percentage of red blood cells with hemoglobin concentration <280 g/L, iron deficiency can be detected.

threshold values vary (28-30 pg), depending on the laboratory and instrument used.

deficient erythropoiesis (Fishbane *et al*., 1997; Mast *et al*., 2002; Brugnara 2003).

which is obtained by an optical cell-by-cell hemoglobin measurement.

previous weeks.

erythropoiesis.

**1.3.1 Siemens**

Thomas, 2002).

(Cullen *et al*., 1999).

transferrin receptor and also tends to be elevated in the presence of increased erythroid activity. It does seem to be a reasonable index of erythropoietic activity (Chiang *et al*., 2002; Tarng & Huang, 2002) and reflects the effect of stimulating bone marrow red cells production, before an increase in reticulocytes is noted and well before the Hb rises; therefore an increase in the sTfR may be the first detectable measure. It is not affected by inflammation (Beerenhout *et al*., 2002) and this reason would make sTfR a more reliable test than serum ferritin.

Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. Interest has been generated in the use of erythrocyte and reticulocyte parameters, available on the modern analysers based on flow cytometry technology.

The modern hematological parameters contribute to the advanced study of the anemia and depend on the technology employed; the debate about other parameters with the same clinical meaning and potential utility as reticulocyte hemoglobin content and percentage of hypochromic red cells is open.


5.2.6. Nitro-Dur System

This type of system is fabricated by heating a hydrophilic polymer, glycerol, and polyvinyl alcohol and then lowering their temperature to develop a polymer gel. Nitrogylcerin and lactose are mixed and dispersed in the polymer, which is solidified at room temperature to form a medicated disc by molding technique. This is assembled into a metallic plastic laminate where the transdermal patch is developed with an adhesive rim. This patch is designed for application to the skin for continuous release of 0.5 mg/cm2 for angina treatment. Poly (isobutylene) or poly (acrylate) adhesive are the polymers used in this method and this polymeric adhesive is spread by solvent casting technique over the area of the drug impermeable membrane (Figure 17) [140]. The marketed formulations are Frandol tape by Toaeiyo/Yamanouchi in Japan—isosorbide dinitrate [141]; Nitro-Dur II by Key in the United States—nitroglycerin, which replaces Nitro-Dur, a first generation patch from the market;Habitrol and Nicotrol—nicotine (smoking cessation) [142]; Minitran—nitroglycerin [143], Testoderm—testosterone (testosterone replacement therapy) [144], and Climara—estradiol (vasomotor system treatment associated with menopause) [145].

**Figure 17.** Nitro-Dur system (adapted from [140]).


**Treatment**

328 Hearing Loss

Zallocchi M, Meehan DT, Delimont D, Askew C, Garige S, Gratton MA, Rothermund-

Franklin CA, Cosgrove D (2009) Localization and expression of clarin-1, the Clrn1 gene product, in auditory hair cells and photoreceptors. Hear Res 255:109-120. Zou J, Luo L, Shen Z, Chiodo VA, Ambati BK, Hauswirth WW, Yang J (2011) Whirlin

Replacement Restores the Formation of the USH2 Protein Complex in Whirlin

Young RW (1967) The renewal of photoreceptor cell outer segments. J Cell Biol 33:61-72. Young RW (1976) Visual cells and the concept of renewal. Invest Ophthalmol Vis Sci 15:700-

Knockout Photoreceptors. Invest Ophthalmol Vis Sci 52:2343-2351.

725.

**15**

Julia Sarant

*Australia*

*The University of Melbourne*

**Cochlear Implants in Children: A Review**

In 1980, the first child in the world was implanted with the single-channel House cochlear implant device (Eisenberg & House, 1982). Children who initially received cochlear implants during this first paediatric clinical trial were quite old compared to current ages (the average age in the first House clinical trial was 8 years, whereas children are now being implanted as young as 6 months of age), and the majority communicated using sign language (Eisenberg & Johnson, 2008). It is now known that implanting older children who do not communicate orally gives little chance of speech perception or spoken language development. In 1985, the first children received a multichannel cochlear implant in Australia (Clark et al., 1987). This clinical trial selected children who had a higher potential for success, including shorter duration of deafness and a commitment to oral communication both at home and in their educational programs. At this time, it was unknown whether the speech processing schemes used with adults who had lost their hearing after developing language (ie. post-lingually deafened) would be appropriate for facilitating the speech perception and language development of young children with immature auditory systems. It is important also to note that the desired outcomes for adults and children differed; while the goal for adults was to improve auditory skills and communication using previously acquired cognitive, spoken language, and social skills, the goal for children was to develop these skills using the auditory information provided by the cochlear implant, having had no useful auditory experience (and therefore presumably no neural development of their auditory system) until they received their cochlear implant. The implantation of children was also highly controversial. For many years, cochlear implantation in children was opposed by the Deaf Community, on the grounds that deafness in children should be considered as a cultural and linguistic difference rather than as a disability that could be remediated by a cochlear implant. Over time, this view has changed such that in 2000, a position paper of the National Association of the Deaf in the U.S. stated that "cochlear implantation is a technology that represents a tool to be used in some forms of communication, and not a cure

It is now well documented that children with severe-profound hearing loss receive significant benefits from cochlear implants in terms of speech perception and language development (Blamey et al., 2006; Geers et al., 2008; Moog, 2002; Nicholas & Geers 2007). Cochlear implants are becoming the standard of care for children with severe-profound hearing loss, with increasing uptake of simultaneous bilateral implants over recent years. There is a large variation in implementation of cochlear implant technology around the

**1. Introduction**

for deafness" (National Association of the Deaf, 2000).


**7. Mental health impacts of noise**

Environmental noise is not believed to be a direct cause of mental illness, but it is assumed that it accelerates and intensifies the development of latent mental disorder. Studies on the adverse effects of environmental noise on mental health cover a variety of symptoms, including anxiety, emotional stress, nervous complaints, nausea, headaches, instability, argumentativeness, sexual impotency, changes in mood, increase in social conflicts, and general psychiatric disorders such as neurosis, psychosis, and hysteria [17, 21].

 This is because it is contrary to the expectation that home is a place of rest and a comfortable, quiet place.

New Yorkers, like citizens in the quietest towns of the country, expect less noise when they close the doors to their apartment and homes. They may willing to deal with the noisy street traffic, crowds, and subways, as they transverse the city but they are less tolerant of noisy intrusions into their homes (Why noise matter chap 2) [3]*.*

It turned out that psychiatric disorders are associated with noise sensitivity, rather than with noise exposure level, and the association was found to disappear after adjustment for the baseline trait anxiety. These and other results show the importance of taking vulnerable groups into account because they may not be able to cope sufficiently with unwanted environmental noise. This is particularly true of children, the elderly, and people with preexisting illnesses, especially depression. Despite the weaknesses of the various studies, the possibility that community noise has adverse effects on mental health is suggested by studies on the use of medical drugs, such as tranquilizers and sleeping pills, on psychiatric symptoms and on mental hospital admission rates. About 1 of 10 people are particularly noise sensitive. These people will become 10% more annoyed by noise than general population [22].

After adjustment of noise-related variables, sociodemographic factors, medical illness, and duration of residence, subjects in the high noise-sensitive (NS) group were more than 2 times more likely to experience depression and insomnia and 1.9 times more likely to have anxiety, compared to those in the low NS group. The levels of noise recognition and psychological discomfort are affected by various factors,

**33**

**Conflict of interest**

*Neighborhood Noise*

individuals [23].

**8. Conclusion**

*DOI: http://dx.doi.org/10.5772/intechopen.92877*

correlated with DA as was noise annoyance [24].

provide national support for noise reduction.

The author declares no conflict of interest.

including individual components (e.g., age and effects of traits) and environmental factors, including contextual aspects and noise parameters (e.g., source, attitude toward noise, and amplitude modulation). Not all people exposed to environmental noise suffer from a disease or health problem, and the effects of noise differ among

Hearing noises above the perceived normal threshold, higher noise sensitivity, and continuous noises were associated with higher levels of displaced aggression (DA). It occurs when a person is provoked, is unwilling or unable to retaliate against the original provocateur, and subsequently aggresses against a seemingly innocent target. Low frequency and high intensity noises were also associated with higher DA scores. DA score was higher in women and in older people living in the neighborhood for a longer time, in people with better education, and in those reporting poorer health. Moreover, low frequency and continuous noises resulted in higher DA. The frequency of hearing noises above the normal threshold was positively

The laws related to noise are increasingly being strengthened, and the actual noise level is decreasing compared to the past with the improvement of building technology and the reinforcement of regulatory standards. However, social problems related to noise have become more serious and are now an important part of the psychoacoustic problems related to the environment. This is also related to the expectation of a better quality of life as a result of the improvement of the economic level and the desire for home comfort. Also, even when the actual noise level is not high compared to other external environments and problems occur between neighbors. Therefore, it means that the problem of neighborhood noise should be discussed not only at the physical level but also at the psychoacoustic aspects, and the individual's sensitivity and cultural difference should be considered. In particular, it needs to be treated more seriously, considering that health problems (especially mental problems) related to neighborhood noise may occur at a lower level than actual measured noise levels. Also, neighborhood noises tend to provoke the existing mental health problems related to noise more easily. Identifying the physiological and psychological effect of environmental noise precisely and making it recognized broadly comprise the essential part of solving the environmental noise problem to create better living environment. The neighborhood noise issues were in part due to the lack of communication between and among neighbors. Lack of contact within the community affected people's perception of the loudness of daily life sounds [6]. Furthermore, intense emotional conflicts between and among some neighbors were also found to be one of the major sources of daily life noise. Even though the neighborhood noise annoyance will always be an issue, we want to be good neighbors and also hope to have good relationship with neighbors. Consideration of neighborhood, social responsibility, and changes in social behavior are important factors in addressing neighborhood noise. In addition, it is necessary to understand a scientific basis for the health impact of noise and to

#### *Neighborhood Noise DOI: http://dx.doi.org/10.5772/intechopen.92877*

including individual components (e.g., age and effects of traits) and environmental factors, including contextual aspects and noise parameters (e.g., source, attitude toward noise, and amplitude modulation). Not all people exposed to environmental noise suffer from a disease or health problem, and the effects of noise differ among individuals [23].

Hearing noises above the perceived normal threshold, higher noise sensitivity, and continuous noises were associated with higher levels of displaced aggression (DA). It occurs when a person is provoked, is unwilling or unable to retaliate against the original provocateur, and subsequently aggresses against a seemingly innocent target. Low frequency and high intensity noises were also associated with higher DA scores. DA score was higher in women and in older people living in the neighborhood for a longer time, in people with better education, and in those reporting poorer health. Moreover, low frequency and continuous noises resulted in higher DA. The frequency of hearing noises above the normal threshold was positively correlated with DA as was noise annoyance [24].


**Conflict of interest**

"The authors declare no conflict of interest."

*Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

*Genetic Variation*

keep on to reduce [111].

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

analysis of EMS mutagenesis.

(*Indica* and *Japonica*) by SNPs genotyping [120].

tion of genetic diversity of rice mutants.

"The authors declare no conflict of interest."

multiplexed SNPs genotyping analysis is necessary to utilize the huge SNPs source for high-throughput genetic test in rice [110]. It will become routine to re-sequence the plants genome with current SNPs platforms as the price of genome sequencing

SNPs genotyping have been applied in many organism including rice [112–114], *Arabidopsis* [115], maize [116], soybean [117] and wheat [118]. The high class order of the rice genome has offered genome-wide SNPs source [119]. Polymorphic loci (5.41 million) were detected between the two main domesticated rice subspecies

A complete map of rice genome builds 6,119,311 SNPs variants for 1529 genome orders. SNPs (213,188) were located in *Indica* and *Japonica* rice. Asian and African rice were established 9595 SNPs [121]. Three thousand rice genomes project [122] submit for rice clustering of aus/boro genotypes. Only 208 accessions are categorized as aus/boro depends upon SNPs markers of 200,000. It is also exposed from further study that aus group was genetically related by 376,000 SNPs markers [123].

The considerable amount of phenotypic variability can be identified by employing highly sophisticated Molecular approaches like SSR and SNPs within the mutant populations. These genetic changes indicated that EMS might be helpful for the development of desired genetic changes in crop plants. It was also recommended that current SSRs and SNPs markers could be suited in further analysis for estima-

The authors acknowledge Higher Education Commission (HEC), Islamabad, Pakistan for providing funds for research activities at Genomics lab, CABB. The authors are thankful to Plant Breeding Division (NIAB) for providing assistance in

**16**


**5.1 Importance of lipoxygenase inhibition for food industry**

Besides their physiological role, plant lipoxygenases are of significant importance to the food industry, since these enzymes have been implicated in the generation of the flavour and aroma in many plant products. For instance, they are responsible for the undesirable 'beany', 'green' and 'grassy' flavours produced during processing and storage of protein products derived from legume seeds (Fukushima, 1994; Robinson et al., 1995) and the development of the stale flavour in beer during storage (Kobayashi et al., 1993). Lipoxygenases also play an important role in the baking industry. They are quite effective as bleaching agents, increase mixing tolerance and improve dough rheology (Nicolas & Potus, 1994; Larreta-Garde, 1995; Cumbee et al., 1997; Borellib et al., 1999).

Freshly refined soybean oil is practically odourless and bland, but "green, grassy, fishy" offflavors may develop quickly if the oil is heated or stored under conditions that expose it to light and oxygen or by contamination with pro-oxidant metals such as copper and iron (Berk, 1992). "Beany" flavour is the principal inconvenience of traditional soymilk and its products (e.g., tofu) and is caused by some ketones and aldehydes, particularly hexanals and heptanals, produced through LOX catalyzed oxidation (Berk, 1992).

Fish lipids are susceptible to oxidation owing to the high levels of polyunsaturated fatty acids (PUFA), even in frozen storage, and this can affect the flavour, texture, taste, aroma and shelf life of fish (Ke & Ackman, 1976). Since the direct interaction between oxygen and highly unsaturated lipids is kinetically hindered (Kanner et al., 1987), the enzymatic initiation of oxidation by enzymes such as lipoxygenase, peroxidases and microsomal enzymes has been gaining favour.

Green tea glazing was shown to improve the storage quality of frozen bonito fillets (Lin & Lin, 2005). In addition, hot water tea extract was shown to suppress the pro-oxidant activities of the dark meat and skin of blue sprat (Seto et al., 2005). Banerjee (2006) proposes that the improvement in the shelf life of fish by green tea polyphenols is at least in part due to inhibition of LOX resulting in delaying oxidation of fish lipids and because of that impregnation of muscle fillets in tea extract by itself or in combination with other natural inhibitors may improve the shelf-life and storage quality of fish fillets.

Besides its function of oxidizing the polyunsaturated fatty acids (linoleic, linolenic and arachidonic), the enzyme may also catalyse the co-oxidation of carotenoids, resulting in the loss of natural colorants and essential nutrients (Robinson et al., 1995). LOX have been implicated in the generation of the flavour and aroma in many plant products, in the decolourisation of pigments and in the potential of compromising the anti-oxidant status (Casey, 1999). In pasta the involvement of LOX in colour loss is demonstrated by positive correlation between the decrease of β-carotene content after pastification and LOX activities in semolina. In addition to this, the hydroperoxidation and bleaching activities of LOX are highly correlated demonstrating that the bleaching might be ascribable to a co-oxidative action by LOX (Borrellia et al., 1999).

During pasta processing in which the maximal pigment degradation by LOX activity occurs (Borrellib et al., 1999), it is shown that externally added β-carotene can act as inhibitor of the LOX-catalysed linoleate hydroperoxidation and an inverse relation between the % of carotenoid loss and the initial carotenoid content in semolina from durum varieties, showing similar LOX activity, was found (Trono et al., 1999).

The complete characterisation of lipoxygenase from pea seeds (*Pisum sativum var. Telephone L.*) gives possibility to avoid destructive influence during food processing and storage (Szymanowska et al., 2009) by the action of this enzyme.