Neeraj Mathur

Correlation of phthalate exposures with semen quality

Phthalates are widely used man-made chemical released in the environment and human exposure is mainly through diet. As the phthalate plasticizers are not covalently bound to PVC, they can leach, migrate or evaporate into the environment and as a result have become ubiquitously contaminants. The present study investigates the correlation, if any, between the phthalate esters (DEP, DEHP, DBP, DMP, DOP) and sperm mitochondrial status, ROS, LPO, SCSA, and sperm quality. The study was conducted in the urban/rural population of Lucknow visiting Obstetrics and Gynecology Department, CSMMU, Lucknow. Semen analysis was performed according to the WHO guidelines while phthalate analysis by HPLC and LPO by spectrophotometer and the sperm mitochondrial status, ROS, SCSA using flow cytometry. The questionnaire data showed no significant difference in the demographic characteristics among the groups. In general, urban population was found to have statistically significant higher levels of phthalate esters than the rural. Further, infertile men showed statistically significant (p<0.05) higher levels of pollutants in the semen than fertile men. A negative correlation between semen phthalate level viz DEHP and sperm quality and positive association with depolarized mitochondria, elevation in ROS production and LPO, DNA fragmentation was established. The findings are suggestive that phthalates might be one among the contributing factors associated with the deterioration in semen quality and these adverse effects might be ROS, LPO and mitochondrial dysfunction mediated.

Lead and cadmium concentration in the seminal plasma of men in the general population: correlation with sperm quality

The concentration of lead and cadmium in the seminal plasma of men in the general population, including fertile and infertile subjects, was measured. Semen samples were categorised as demonstrating oligospermia (sperm concentration < 20 x 10(6)/ml), asthenospermia (<50% motilesperm), oligoasthenospermia (a combination of the two criteria), and azoospermia (no sperm). An increase in lead and cadmium levels was observed in infertile men and there was a significant negative correlation of cadmium and lead semen concentration with sperm motility and sperm concentration in oligoasthenospermic men.

The effect of smoking and eating habits on DNA damage in Indian population as measured in the Comet assay

This study was undertaken with the aim of assessing the status of DNA damage in a normal healthy Indian population. The 62 male volunteers in this study belonged to the smoking, non-smoking, vegetarian and non-vegetarian categories, were well educated and aged between 23 and 57 years. The data revealed significant differences in the extent of DNA damage in the smokers versus non-smokers as well as between the vegetarians and non-vegetarians. A significant difference was also observed amongst the different groups of smokers depending on the extent of smoking. An age-dependent effect in DNA damage was also observed. This preliminary study has, for the first time, revealed differences in the extent of DNA damage in the normal Indian population depending on their eating and smoking habits as well as age.

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay.

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.

Adverse health effects of pesticides in agrarian populations of developing countries.

Developing countries use only 20% of the worlds agrochemicals, yet they suffer 99% of deaths from pesticide poisoning. Pesticide poisoning is a significant problem in developing countries primarily because of unsafe pesticide application and handling practices. Safety is further exacerbated by the illiteracy and poverty that prevails in most farming communities of developing countries. Pesticides classified as being extremely or highly hazardous by FAO and WHO, including those banned by other countries, continue to be used in developing countries. Many farmers in developing countries continue to be exposed to pesticides from either storing them in or near their residences, or from inadequate or unsafe application or handling practices. Farming populations exposed to pesticides suffer from several health problems, primarily neurological abnormalities, respiratory ailments, and reproductive, endocrinological, and dermal problems. In developing countries, the scientific literature (including the Indian Institute of Toxicology Research, India) have taken the initiative to monitor health problems resulting from pesticide exposure in agrarian communities. The welfare fund for agricultural laborers could institute a special program for pesticide applicators in developing countries. The primary need, currently, in such countries is creation and implementation of sound national policies to effectively articulate appropriate guidelines for managing farm pest control activities. Such policies should be aimed at both limiting pesticide exposure and usage, but doing so without damaging the yields of food production. If such steps are taken, it is fully expected that the incidence of adverse health consequences for agrarian populations from pesticide toxicity will decrease, and the health of farmers improve.

Clinical, biochemical and neurobehavioural studies of workers engaged in the manufacture of quinalphos.

59 workers exposed to different chemicals during the manufacture of quinalphos, an organophosphate pesticide (OP) and 17 control subjects were studied. Despite similar blood acetylcholinestarase (AChE) levels in both the exposed and control subjects, a significant number of exposed subjects had altered plantar and ankle reflexes. Higher nervous functions such as memory, learning and vigilance were also found to be affected in these subjects. These findings were attributed to chronic low dose combined exposure to different chemicals used/formed in the manufacture of quinalphos. The study raises the doubt that monitoring of AChE alone among subjects engaged in the manufacture of OP pesticides may not be an adequate safeguard as regards to their health.

Environmental and experimental exposure of phthalate esters: The toxicological consequence on human sperm

Rapid industrialization and urbanization release several chemicals such as phthalates into the environment and cause adverse effects on reproductive system, mainly endocrine disruption, testicular injury and decline in semen quality in humans. There are no reports in extrapolating of the epidemiological data with in vitro findings. Our study show the correlations between in vivo studies and in vitro data for the effect of phthalate esters. Healthy human males, in the age group 21 to 40 years, visiting Chhatrapati Sahuji Maharaj Medical University (CSMMU), Lucknow, as part of infertility investigation, were recruited as volunteers. Semen analysis was performed according to the WHO guidelines. Phthalate esters were analyzed by high-performance liquid chromatography (HPLC) and cell viability by MTT assay. In the in vitro studies, sperms were exposed to highest concentration in semen samples (5—10 times higher) for a period ranging between 30 min and 96 hours. An inverse relationship with sperm motility in epidemiological studies was concurrent by significant dose-and time-dependent decrease in the sperm motility under in vitro environment after 12-hour exposure. Cytotoxicity was observed only with the highest concentration after 96 hours of exposure. There are a significant correlation between phthalate ester diethylhexyl phthalate, di-n-butyl phthalate (DEHP and DBP) and sperm motility both in vitro and in vivo conditions. Additionally, in vitro experiments conducted not only adjunct to the existing in vivo data but also specify the effect of specific toxicants (DEHP and DBP) on sperm motility and viability. Results show the decrease in motility of sperms under in vitro conditions at the maximum range of in vivo measured levels and 5- or 10-folds higher to that found in human semen samples.

Evidence for increased cytochrome P450 1A1 expression in blood lymphocytes of lung cancer patients

To develop blood lymphocyte cytochrome P450 1A1 (CYP1A1) expression as a surrogate for monitoring tissue expression for polycyclic aromatic hydrocarbon (PAH) induced toxicity, the present study attempted to characterize CYP1A1 mRNA expression and its associated catalytic activity in freshly prepared blood lymphocytes isolated from healthy controls and patients suffering from tobacco induced lung cancer. Human blood lymphocytes were found to express CYP1A1 mRNA and significant activity of 7-ethoxyresorufin-O-deethylase (EROD). Significant increase in the activity of EROD and CYP1A1 mRNA was observed in blood lymphocytes isolated from patients suffering from lung cancer. Further, controls with variant genotypes of CYP1A1 (Msp1 or Ile/Val polymorphism) exhibited significant increase in the enzyme activity associated with an increase in CYP1A1 mRNA expression when compared to the controls with wild type genotype. Patients with variant genotypes of CYP1A1 also exhibited much greater increase in the blood lymphocyte CYP1A1 mRNA expression and EROD activity when compared to controls or patients with wild type genotype. Our data thus provides evidence of CYP1A1 expression in freshly isolated blood lymphocytes and differences in reactivity in individuals with variant genotypes of CYP1A1, suggesting that blood lymphocyte CYP1A1 expression profile could help in identifying individuals at risk to environment induced lung cancer.

Association of functionally important polymorphisms in cytochrome P4501B1 with lung cancer.

In the present study, genotype and haplotype frequencies of four polymorphisms of cytochrome P450 1B1 (CYP1B1) that cause amino acid changes (Arg-Gly at codon 48, Ala-Ser at codon 119, Leu-Val at 432 and Asn-Ser at codon 453) were studied in 200 patients suffering from lung cancer and equal number of controls. A significant difference was observed for the distribution of variant genotypes of CYP1B1Arg48Gly and Ala119Ser polymorphisms (CYP1B1*2) in cases when compared to the controls. No significant difference was observed for the distribution of variant genotypes of CYP1B1Leu432Val (CYP1B1*3) and CYP1B1Asn453Ser (CYP1B1*4) polymorphism. When the four SNPs were analyzed using a haplotype approach, SNPs at codon 48 (Arg48Gly) and codon 119 (Ala119Ser) exhibited complete linkage disequilibrium (LD) in all the cases and controls. Significant differences in the distribution of the three haplotypes (G-T-C-A, G-T-G-A and G-T-C-G) were observed in the cases when compared to controls. Tobacco use in the form of smoking as well as chewing was found to significantly increase the risk of lung cancer in patients by interacting with CYP1B1Ala119Ser genotypes demonstrating the role of gene-environment interaction in lung cancer. Further, the risk of lung cancer increased several fold in the patients carrying the genotype combinations of CYP1B1Ala119Ser and CYP1B1Leu432Val with GSTM1, a phase II enzyme suggesting the importance of gene-gene interactions in enhancing the susceptibility to lung cancer.

A Study on the Association of Cytochrome-P450 1A1 Polymorphism and Breast Cancer Risk in North Indian Women

Cytochrome P-450 1A1 (CYP1A1) is involved in the 2-hydroxylation of estrogens and mammary carcinogens into 2-hydroxy catechol metabolites. Many commonly occurring single nucleotide polymorphism (SNP) are reported in CYP1A1 in various populations that include, isoleucine to valine substitution at 462 codon in heme binding region in exon 7 (A to G transition at position 2455; M2), threonine to asparagine substitution at codon 461 (C to A transversion at position 2453; M4), T to C transition at 3801 position (M1) and T to C transition at position 3205 (M3) in 3′ non-coding region. Epidemiological studies have shown inconsistent patterns between CYP1A1 polymorphism and breast cancer risk among various populations. Most of the studies have shown significant association between CYP1A1 genotype polymorphism and breast cancer risk. The present investigation was therefore undertaken to investigate the association of M1, M2, M3 and M4 polymorphisms and their subsequent contribution in premenopausal and postmenopausal women with breast cancer risk in north Indian women. Genomic DNA was isolated from case controls and breast cancer patients, specific segments of genomic DNA were amplified and restriction fragment length polymorphism (RFLP) was performed. CYP1A1 expression and catalytic activity were also assessed in premenopausal and postmenopausal case controls and patients. Polymorphism at M1, M2 and M4 alleles was detected and odds ratio for W/M1 and␣M1/M1 was calculated as 1.07 (95% CI, 0.59–1.87) and 0.74 (95% CI, 0.28–1.96) respectively. Odds ratio for W/M1 and M1/M1 alleles in premenopausal and postmenopausal women was 1.09 (95% CI, 0.45–2.49)/0.62 (95% CI, 0.10–2.66) and 1.60 (95% CI, 0.60–4.22)/1.06 (95% CI, 0.22–7.33) respectively. Odds ratio for W/M4 and M4/M4 allele was 1.20 (95% CI, 0.65–2.24)/4.55 (95% CI, 0.44–226.2) and 0.96 (95% CI, 0.36–2.64)/4.51 (95% CI, 0.23–273.0) respectively in total and premenopausal women. In postmenopausal women odds ratio was calculated as 1.16 (95% CI, 0.45–2.94) for M4/W but it could not be detected for M4/M4 since this genotype was not found in any postmenopausal case controls. Odds ratio for W/M2 genotype was calculated 0.57 (95% CI, 0.28–1.02), 1.06 (95% CI, 0.40–2.47) and 0.33 (95% CI, 0.12–0.89) respectively for total, premenopausal and postmenopausal women, however, in any group the odds ratio for M2/M2 could not be detected as M2/M2 genotype was not found in breast cancer patients. Polymorphism at M1 and M4 alleles was not found significantly associated with breast cancer risk and only wild type genotype was found in case controls and patients for M3 allele. Lack of protective association between CYP1A1 M2 genotype was also observed, however, in postmenopausal women a significant protective association with breast cancer risk was found (odds ratio, 0.33; 95% CI, 0.12–0.89; P-value 0.03). Similarly, no significant alteration in CYP1A1 expression and catalytic activity was observed in wild type and variant genotypes both in premenopausal and postmenopausal patients as compared with their respective controls. The results obtained from the present investigation thus suggest that probably CYP1A1 (M1, M2, M3, and M4) polymorphism alone does not play a significant role in the breast cancer risk in north Indian women.