D. K. Saxena

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.

Comparative toxicity of trivalent and hexavalent chromium: Alterations in blood and liver

The effects of trivalent and hexavalent chromium compounds on rabbits were studied with a view toward investigating the toxic potentials of two different forms of chromium to which industrial workers or miners might be exposed. While both forms of chromium altered the levels of certain important chemical constituents of blood and serum and produced significant morphological changes in the liver, the hexavalent form induced a greater effect. The accumulation of the metal was also higher in animals exposed to chromium in oxidation state 6 than in those exposed to metal in state 3.

Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: role of ROS generation.

Exposure to benzene, toluene and xylene in the human population may pose a health risk. We tested a working hypothesis that these test chemicals cause cellular toxicity to a non-target organism, Drosophila melanogaster. Third instar larvae of D. melanogaster transgenic for hsp70, hsp83 and hsp26 and Oregon R(+) strain were exposed to 1.0-100.0 mM benzene, toluene and xylene for 2-48 h to examine the heat shock proteins (hsps), ROS generation, anti-oxidant stress markers and developmental end points. The test chemicals elicited a concentration- and time-dependent significant (p<0.01) induction of the hsps in the exposed organism in the order of hsp70>hsp83>or=hsp26 as evident by beta-galactosidase activity after 24 h. RT-PCR amplification studies in Oregon R(+) larvae revealed a similar induction pattern of these genes along with hsp60 in the order of hsp70>hsp60>hsp26>or=hsp83. Under similar experimental conditions, a significant induction of ROS generation and oxidative stress markers viz. superoxide dismutase, catalase, glutathione S-transferase, thioredoxin reductase, glutathione, malondialdehyde and protein carbonyl content was observed. Sub-organismal response was propagated towards organismal response i.e., a delay in the emergence of flies and their reproductive performance. While hsp70 was predominantly induced in the organism till 24 h of treatment with the test chemicals, a significant or insignificant regression of Hsp70 after 48 h was concurrent with a significant induction (p<0.01) of hsp60>hsp83>or=hsp26 in comparison to the former. A significant positive correlation was observed between ROS generation and these hsps in the exposed organism till 24 h and a negative correlation between ROS generation and hsp70 in them after 48 h indicating a modulatory role of ROS in the induction of hsps. The study suggests that among the tested hsps, hsp70 may be used as an early bioindicator of cellular toxicity against benzene, toluene and xylene and D. melanogaster as an alternative animal model for screening the risk posed by environmental chemicals.

Manganese-induced behavioral dysfunction and its neurochemical mechanism in growing mice

Abstract— Suckling mice were exposed to manganese from birth indirectly through their mothers and then directly through drinking water after weaning. The growth and development of these mice and their age‐matched controls were almost identical. Motor activity of offspring measured at 30‐day intervals showed a significant increase at 60 and 90 days in manganese‐treated mice compared to controls. Increased motor activity was associated with significant elevation in the levels of dopamine and norepinephrine in the corpus striatum of treated mice. The levels of striatal tyrosine, homovanillic acid and manganese were also significantly increased in mice after manganese exposure. Thus an animal model of early manganese poisoning has been developed with a possible role of striatal amines in the production of behavioral dysfunction in the treated mice. Implications of these findings are discussed in relation to the manifestations of the psychiatric phase of early manganese poisoning in man.

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.

Induction of hsp70 in transgenic Drosophila: biomarker of exposure against phthalimide group of chemicals

The expression of stress genes is suggested to be a potentially sensitive indicator of any chemical or physical assault. This led us to explore the possibility of using expression of one of the major stress genes, hsp70, in Drosophila as a biomarker against phthalimide group of chemicals, which may accordingly provide an early indication of exposure to these hazardous chemicals. We exposed third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9) to different concentrations of the test chemicals (Captan, Captafol and Folpet) for various time intervals (2-48 h) to evaluate expression of hsp70 by X-gal staining, ONPG assay and whole organ in situ immunohistochemistry. The study was further extended to examine the effect of the said chemicals on development of the organism and tissue damage occurring in them, thus raising the possibility of evaluating comparative deleterious effect inducing potential of the test chemicals. Our results showed a strong hsp70 expression in the Captafol-exposed larvae followed by weaker expression in Captan- and Folpet-treated larvae. The effect was further reflected on development as revealed by a delay in emergence of the flies by 3 days in 200 ppm Captafol-exposed group. Hsp70 was found not to be induced at 0.0002 ppm Captafol and at 0.002 ppm Captan and Folpet. The present study suggests that (a). hsp70 induction is sensitive enough to be used as a biomarker against phthalimide group of chemicals, (b). amongst the three test chemicals, Captafol is the most deleterious compound followed by Captan and Folpet, (c). 0.0002 ppm for Captafol and 0.002 ppm for Captan and Folpet, respectively, can be regarded as no observed adverse effect level (NOAEL).

Evaluation of the No Observed Adverse Effect Level of Solvent Dimethyl Sulfoxide in Drosophila melanogaster.

Dimethyl sulfoxide, a solvent commonly used in toxicological studies, was investigated for its cytotoxic potential and its effect on development and reproductive performance in transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9. Various concentrations (0.0, 0.1, 0.3, 0.5, 1.0, 2.0, and 3.0%) of the solvent were mixed with food and fed to the flies and larvae. The toxic effects were studied by examining hatchability, emergence, fecundity, reproductive performance, and hsp70 expression by means of in situ β-galactosidase staining in the tissues of third-instar larvae and in the reproductive organs of male and female flies of the strain. Our results showed that dimethyl sulfoxide at and above 0.5% of dietary concentration evoked cytotoxicity, as evidenced by hsp70 expression in the larval tissues, and also impaired the development and reproductive performance of the flies. This study suggests that the no observed adverse effect level of dimethyl sulfoxide is 0.3% of dietary concentration for developmental toxicity, reproductive toxicity, and cytotoxicity studies in Drosophila melanogaster.

Embryotoxicity of orally administered chromium in mice: Exposure during the period of organogenesis

Administration of chromium (VI)(250, 500 and 750 ppm as potassium dichromate) via drinking water during organogenesis (days 6-14 of gestation) in mice revealed embryo- and fetotoxic effects. Reduced fetal weight, retarded fetal development, number of fetuses (live and dead) per mother and high incidences of dead fetuses and resorptions in treated mothers in the highest dosed group were evident. No significant gross structural abnormalities were observed in any of the fetuses of chromium (VI)-treated mothers. Significant incidences of reduced ossification were found in the highest dosed group. Chromium levels were increased in a dose-dependent manner in maternal blood, placenta and fetuses. The present study suggests a risk to the developing embryo if the mother is exposed to a sufficiently high concentration of chromium (VI) through drinking water during the period of organogenesis.

Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead

Groups of rats were exposed simultaneously to manganese chloride (3 mg Mn2+/ml water) through drinking water and lead acetate intraperitoneally at dosages of 5.0, 8.0 and 12.0 mg Pb2+/kg daily for a period of 14 days. The magnitude of changes in the behavioral pattern, contents of biogenic amines and accumulation of lead in the brain of rats simultaneously exposed to the two metals was significantly greater than observed in rats after exposure to either of the metals alone. A definite dose-response relationship was, however, noticed only with the changes in the motoractivity, norepinephrine, 5-hydroxytryptamine levels and in the accumulation of lead in rats simultaneously exposed to manganese and lead. The lowering in the contents of norepinephrine after combined treatment was found to be related with the decrease in the motoractivity in the rats. The exact role of depression in the levels of dopamine and 5-hydroxytryptamine in inducing marked impairment in learning ability and increased aggressive behavior in rats after the combined exposure to manganese and lead could not be ascertained. The overall analysis of the data indicated that the simultaneous exposure to manganese and lead, particularly with highest dose of the latter, may produce serious derangements in the behavioral pattern and levels of biogenic amines in the brain of rats.