Jasna Kniewald

Disorders of male rat reproductive tract under the influence of atrazine

The effects of atrazine exposure on testicular sperm number, epididymal sperm number and motility and α‐glucosidase activity in the epididymis were studied in Fischer rats. Histological changes in the testicular tissue were followed by light and electron microscopy. Groups of adult animals were treated i.p. with 60 and 120 mg atrazine kg−1 body wt. twice a week over 60 days. The results indicate a decrease in the body weight and relative weights of pituitary and ventral prostate vs control, measured on the last day of treatment in both treated groups. Testicular sperm number (expressed as number of sperm per 500 Sertoli cells) in atrazine‐treated groups increased with the treatment time due to the reduced sperm motility. Therefore atrazine treatment provoked a significant decrease in sperm number and motility in epididymis, measured after the last day of treatment. α‐Glucosidase activity in the epididymis, after the last day of treatment, showed a decrease in both treated groups vs control values. Histological analysis of testicular tissue from treated rats showed the cell disorganization and cell clusters together with spermatocytes. Electron microscopy presented differently vacuolated cytoplasm, collagen fibre was reduced, Leydig cells were of irregular shape with unequal form and cisternae of rough endoplasmic reticulum were accentuated and softly widened. In Sertoli cell cytoplasm, atrazine treatment provoked degenerative changes. According to the results obtained, it is evident that atrazine exerted morphological changes and a toxic effect on sperm and their motility. Copyright

Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence

The inhibitory influence of atrazine and deethylatrazine on testosterone metabolism in male rat anterior pituitary and hypothalamus were studied under in vivo and in vitro experimental conditions. In vivo strong influence of atrazine (12 mg/100 g by wt. daily during 7 days) on 5 alpha-R, 3 alpha- and 17 beta-HSD activities was detected in the anterior pituitary. This dose provokes a significant increase in the weight of the pituitary gland, with hyperemia and hypertrophy of chromophobic cells with vacuolar degeneration. In vivo treatment of male rats with the same dose of deethylatrazine markedly inhibited 5 alpha-R activity in the anterior pituitary. The rate of 5 alpha-R activity inhibition in the anterior pituitary was the same after in vivo treatment with atrazine (37.3%) as with deethylatrazine (33.9%). This could suggest that the mechanism of inhibition of deethylatrazine is similar to that of atrazine. In vitro atrazine or deethylatrazine addition into the incubation medium significantly (P less than 0.01) inhibited 5 alpha-R, 3 alpha- and 17 beta-HSD activities in the anterior pituitary. The inhibition of 5 alpha-R activity was marked more by atrazine than deethylatrazine, while 3 alpha- and 17 beta-HSD activities were inhibited at the same rate. In vivo treatment with the same dose of atrazine or deethylatrazine (12 mg/100 g by wt daily 7 days) significantly inhibited (P less than 0.01) 5 alpha-R and 17 beta-HSD at the male rat hypothalamic level. 3 alpha-HSD activity inhibition was not significant for either compound. The in vitro addition of deethylatrazine was much more effective (P less than 0.01) in inhibiting 5 alpha-R, 3 alpha- and 17 beta-HSD in male rat hypothalamus than atrazine. In spite of this, deethylatrazine seems to be less toxic in in vivo experiments due to its higher polarity and faster biodegradation.

Reversibility of the inhibitory effect of atrazine and lindane on cytosol 5 alpha-dihydrotestosterone receptor complex formation in rat prostate.

Once entering the bloodstream, most toxic substances, including pesticides, can reach organs involved in the reproductive system. They can cross the placenta, as well as the brain barrier, posing various risks to the reproductive processes. The organochlorine insecticide lindane and the s-triazine herbicide atrazine produce changes in hormone-dependent reactions in the rat hypothalamus, anterior pituitary, and prostate. Lindane also causes histological and biochemical alterations in the rat testis. In vivo treatment with atrazine produces a markedly inhibitory influence of 5{alpha}-dihydrotestosterone - receptor complex formation in rat prostate cytosol. Therefore, the aim of this study was to investigate whether such changes in the crucial step in the reproductive process are reversible. A parallel investigation using lindane was also undertaken.

Effect of pesticides on oestradiol-receptor complex formation in rat uterus cytosol.

The effect of gamma-1,2,3,4,5,6-hexachlorocyclohexane (lindane), 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) and 2-methylthio-4,6-bis-isopropylamino-s-triazine (prometryne) on the formation of a specific oestradiol-receptor complex in the rat uterus cytosol has been examined in vitro and in vivo. Both in vitro and in vivo, the pesticides significantly (P < 0.001) inhibited the formation of the complex in the rat uterus cytosol. The decrease in the number of free specific binding sites on the receptors was determined. The affinity of binding was not modified under the influence of pesticides, and the Kd value was of the same order of magnitude (10(-9) M). The inhibition was found to be fully non-competitive.

Effects of s-triazine herbicides on hormone-receptor complex formation, 5α-reductase and 3α-hydroxysteroid dehydrogenase activity at the anterior pituitary level

Abstract Experiments were performed to study the effects of s-triazine herbicides, atrazine ∗ and prometryne, on 5α-dihydrotestosterone (5α-DHT) receptor complex formation and on the activity of the 5α-reductase and 3α-hydroxysteroid dehydrogenase system in the anterior pituitary of male rats. By sucrose density gradient separation, it was found that in the presence of 0.4 mmol of atrazine or prometryne, 5α-DHT binding to receptor proteins in pituitary tissue was decreased by 27% and 17%, respectively. In in vitro experiments, the addition of atrazine or prometryne decreases the conversion of testosterone to 5α-DHT and the conversion of 5α-DHT to 5α-androstan-3α,17β-diol (3α-diol) in the anterior pituitary. The concentration in the range of 0.6 to 12 mmol of both herbicides, inhibited the 5α-reductase and 3α-hydroxysteroid dehydrogenase activity from 7–92%. in vivo subcutaneous (s.c.) administration of atrazine and prometryne reduced the 5α-reductase activity in the anterior pituitary. A single dose (0.1 mg/100 g b.w.) of atrazine decreases the amount of the 5α-reduced metabolite by 34%, while the same dose injected twice or a double dose (0.2 mg/100 g b.w.) inhibited by 46%. A single dose of prometryne (0.1 mg/100 g b.w.) does not affect the enzymic activity, while two injections of a single dose or a single injection of a double dose (0.2 mg/100 g b.w.) decreased the 5α-reductase activity by 17%.

Lindane-induced cytotoxicity and the role of vitamin E in Chinese hamster ovary (CHO-K1) cells

Lindane, a toxic insecticide from the persistent organic pollutants (POP’s) group, may act as an endocrine disrupter affecting crucial tissues of reproductive system. In this study a Chinese Hamster Ovary cell line (CHO-K1) was applied to assess the potential of lindane cytotoxicity at the cellular level. The methods of Trypan blue exclusion, MTT and Kenacid blue assays were used to assess cytotoxicity and confirmed a decrease in the number of viable CHO-K1 cells at 34.4–344 μM lindane during 24, 48 and 72 hours of exposure. The cell proliferation tests showed significant inhibition (p < 0.025–0.001 vs control) and a progressive increase in toxicity with increasing lindane concentrations. Corresponding IC50 values were determined with each applied method. After 72 h of lindane exposure, IC50 values were 184 μM according to the Trypan blue method and 272 and 256 μM with the Kenacid blue and MTT assays, respectively. Morphological changes induced by the cytotoxicity of lindane were followed by the fluorescence microscopy and only necrotic cells were detected. Vitamin E (25 and 50 μg/mL) was used for protection of ovarian cells against lidane-induced oxidative stress damage, and lipid peroxidation was postulated as a possible mechanism of lindane toxicity. The viability of cells pre-incubated with vitamin E was significantly enhanced (up to p < 0.025) compared to the results observed in cells exposed to lindane only, but vitamin E treatment could not prevent complete lindane-induced cytotoxicity. Results suggest that vitamin E may exert a slightly protective role in cell defense against lipophilic pro-oxidant xenobiotics such as lindane.

A new approach to the study of hormone-protein interaction using the microcalorimetric method

Binding enthalpies of various hormones to bovine serum albumin (BSA) and human serum albumin (HSA) in 50 mM phosphate buffer, pH 7.4, at 37 degrees C have been determined by direct microcalorimetry. The observed enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to BSA were found to be -13.24 plus or minus 0.11 -10.31 plus or minus 0.02, -2.37 plus or minus 0.46, -17.64 plus or minus 0.32 and -17.14 plus or minus 0.36 kcal/mol of hormone, respectively. under the same experimental conditions the enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to HSA were found to be -23.94 plus or minus 0.32, -18.88 plus or minus 0.49, -11.14 plus or minus 0.02, -9.88 plus or minus 0.14 and -20.85 plus or minus 0.39 kcal/mol of hormone, respectively.

PCB 77 action in ovary cells--toxic effects, apoptosis induction and cell cycle analysis.

Abstract Context: PCB 77 (3,3′,4,4′-tetrachlorobiphenyl), a non-ortho congener with planar configuration, has been identified as potential endocrine disrupter capable to increase the risk of reproductive and developmental failure. Objective: In the present study, in vitro PCB 77 toxic potential, apoptosis induction and cell cycle alterations were investigated to reveal direct toxic effects on ovarian cells. Methods: Chinese Hamster Ovary (CHO-K1) cell line was selected as a model system and decreased cell viability was confirmed by application of four bioassays. Cellular morphology and quantitative analysis of apoptotic, necrotic and viable cells were determined with fluorescent microscopy and cell cycle phase distributions by measuring DNA content using flow cytometry. Results: We have indicated Trypan blue exclusion assay as the most sensitive for quantifying cytotoxicity of PCB 77 in terms of IC50 values, while the results obtained by other methods pointed to a possible localized effect on the lysosomes/endosomes (Neutral red), compromised intracellular metabolic processes (MTT) and possible interferation with the rate of protein synthesis (Kenacid blue). The loss of cell viability, as a consequence of treatment with 10–100 μM PCB 77, fundamentally was due to induction of apoptosis with observed common series of specific morphological changes characteristic to apoptotic phenomenon. The level of alterations of normal cell cycle progression was low without significant changes at analyzed time intervals. Conclusion: These results indicate toxic outcomes of PCB 77 at ovarian cellular level with regard to potential direct adverse effects to female reproductive system.

Atrazine toxicity in male rat reproductive processes

Herbicide atrazine (A) effect on reproductive processes in male rat has been examined at anteroir pituitary (AP), testis (T), prostate (P) and seminal vesicles (SV). In the present study A in corn oil was administred to 90 days old rats i.p. at 120 mg/kg b.w./each 72h/60 days. Exposure of rats had induced changes in LH and FSH synthesis, changes of spermatogenesis and structural changes at all selected tissues. LH and FSH were detected by immunohistochemical method, structural changes by light and electron microscopy, while spermatogenesis has been followed by measuring concentration, total number, motility and progressive motility of sperms. A-males in AP have decreased number of immunoreactive cells for LH 25%, while FSH increased for 70%. In T control FSH is dominantly present on the perifery and is concentrated with the spermatocytes and spermatogonia. In A-rats FSH presence is within the whole irregular organized seminiferous tubules. Histochemistry of P tissue shows the tubular alveolar gland with large irregular cavities in A-rats. Our previous research on DHT-receptors in prostate showed the reduction of DHT specific binding sites on receptor molecules. At SV evidently mucosal folds are more tighty packed in A-rats vs. control. The weight of SV in A-rats was much lower and a typical atrophy of the gland is present. Inhibition of spermatogenesis in A-treated rats was in sperm count from 14.3x106 per ml vs. 17.06106 in controls. At the same time the motile sperm count has been reduced from 43% on 35%, respectively.

Calorimetric approach to the study of 5α-dihydrotestosterone and estradiol receptors in rat hypothalamus cytosol

Abstract The hypothalamus and pituitary are the sites through which androgen and estrogen hormones perform the feedback control of gonadotropic mechanism. The microcalorimetric method was used to study the specific reaction between 5α-dihydrosterone (5α-DHT) and Estradiol (E 2 ) and their receptors in the hypothalamus of male and female rats at the age of 28 d. In order to compare binding affinity, experiments were performed at 4° and 37°C. Molar enthalpy changes for E 2 at 37°C are −43.96 ± 0.453 kcal/mol for female and −25.04 ± 0.222 kcal/mol for male rats, respectively. At 4°C molar enthalpy changes are approximately 50% higher for female and 25% higher for male rats. For 5α-DHT molar enthalpy changes at 37°C are −26.67 ± 1.221 kcal/mol for female and −18.60 ± 0.512 kcal/mol for male rats. At 4°C increase for female rats is over 60% and for male rats 40%. The effect of sex difference is much better expressed for E 2 than for 5α-DHT. Possible binding of E 2 and 5α-DHT to hypothalamus receptors in the female and male rats could be in some connection with the mechanism in regulation of gonadotropic feedback system. Ratios of molar enthalpy changes for E 2 : 5α-DHT are 2:1 in females and 1:1 in males, at 4°C. Difference between these ratios could be an answer for the physiological sex difference.