Effects of the fungicide prochloraz on xenobiotic metabolism in rainbow trout: in vivo induction

1. Rainbow trout were dosed with prochloraz by i.p. injection of sprayed food pellets. Cytochrome P-450, two P-450-dependent activities, and two conjugase activities were measured in vitro in microsomal or cytosolic fractions. 2. Prochloraz increased cytochrome P-450 in liver, intestine, and pyloric caeca: maximum response occurred at 30-100 mg/kg i.p. In cold conditions, this increase persisted for more than 8 days after injection. 3. Hepatic 7-ethoxycoumarin-O-dealkylase (ECOD) and 7-ethoxyresorufin-O-dealkylase (EROD) were inhibited by prochloraz except in one assay in warm water where they increased. In intestine and pyloric caeca, ECOD and EROD were not detected, even when cytochrome P-450 was increased. 4. UDP-glucuronosyltransferase (1-naphthol as substrate) was unchanged or inhibited after prochloraz dosing. 5. Glutathione-S-transferase (o-dinitrobenzene as substrate), was unchanged or inhibited by prochloraz. 6. The measured level of enzymic activities was the result of induction and inhibition by prochloraz residues. Variations in basal activities and perhaps in prochloraz interactions were due to temperature acclimatization.     

Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz.

The commonly used imidazole fungicide prochloraz was tested for antiandrogenic effects in vitro and in vivo. Prochloraz, but not the metabolites 2,4,6-trichlorophenoxyacetic acid or 2,4,6-trichlorophenol, inhibited the R1881-induced response in an androgen receptor reporter gene assay. In the Hershberger assay, prochloraz exposure at all dose levels (50, 100, and 200 mg/kg) given orally to castrated testosterone (T)-treated males markedly reduced weights of ventral prostate, seminal vesicles, musc. levator ani/bulbocavernosus, and bulbourethral gland. These effects were accompanied by an increase in LH and a reduction of the T(4) and TSH level. The effects on seminal vesicles, LH, T(4), and TSH were also evident in intact prochloraz-exposed young adult rats. Body weights were unaffected whereas liver weights were increased in prochloraz-treated animals. Changes in androgen-regulated gene expression were determined in ventral prostates by real-time RT-PCR. A pronounced decrease of ornithin decarboxylase and PBP C3 mRNA levels was observed for both prochloraz and flutamide. These results indicate that prochloraz antagonizes the peripheral androgen receptors resulting in decreased growth of androgen-dependent tissues and that it antagonizes central androgen receptors blocking the negative feed-back mechanism of testosterone resulting in increased LH secretion from the pituitary. The antiandrogenic effects of prochloraz were in many ways qualitatively comparable, although weaker, to the effects of flutamide. However, differential effects on levels of FSH, T(4), and TSH indicate that other modes of action apart from the pure AR antagonism might play a role in vivo.     

Influence of environmental temperature on the induction of xenobiotic metabolism by beta-naphthoflavone in rainbow trout, Salmo gairdneri

The response of hepatic microsomal cytochrome P-450 monooxygenase and UDPglucuronosyltransferase to a common inducer, beta-naphthoflavone, was investigated in rainbow trout acclimated to 5 or 17 degrees C. The hepatic microsomal 7-ethoxycoumarin-O-deethylase, 7-ethoxyresorufin-O-deethylase, and benzo[a]pyrene hydroxylase activities, measured at 5 or 17 degrees C, were induced at both acclimation temperatures. Maximum response of 7-ethoxyresorufin-O-deethylase and 7-ethoxycoumarin-O-deethylase activities to beta-naphthoflavone was obtained with doses of 5 mg/kg and above, both in warm- and cold-acclimated trout. In warm-acclimated trout the maximum degree of induction of cytochrome P-450-dependent activities was already evident on the day following treatment with beta-naphthoflavone (100 mg/kg), whereas these activities reached maximum values after 3 days in cold-acclimated fish. Furthermore, the maximum induced values of 7-ethoxyresorufin-O-deethylase and benzo[a]pyrene hydroxylase activities were higher in cold-acclimated than in warm-acclimated fish. beta-Naphthoflavone treatment caused a slight but significant increase (1.6-fold) in UDPglucuronosyltransferase activity in warm-acclimated fish when measured at 5 or 17 degrees C, whereas in cold-acclimated fish a significant elevation was seen only when the activity was determined at environmental temperature (5 degrees C).     

Metabolic fate of 2,4-dichloroaniline, prochloraz and nonylphenol diethoxylate in rainbow trout: a comparative in vivo/in vitro approach.

The metabolism and distribution of 2,4-dichloroaniline (2,4-DCA), prochloraz and 4-n-nonylphenol diethoxylate (NP2EO) were investigated in vivo and in vitro in rainbow trout (Oncorhynchus mykiss). Each compound was administered p.o. (10 mg/kg wet weight) and urine was collected during 48 h (2,4-DCA, prochloraz) or 72 h (NP2EO). Fish were sacrificed, the gall bladder was excised and radioactivity was measured in tissues, viscera and carcasses. Metabolic profiles were performed by radio-HPLC and when possible metabolites were identified by LC/MS. For comparison, the biotransformation of these xenobiotics was also investigated in freshly isolated hepatocytes. The metabolic pathways of 2,4-DCA have been identified leading to the glucuronide conjugate (in vivo) and to the glucuronide conjugate and the hydroxylamine metabolite (in vitro). This difference highlights the usefulness of the hepatocyte system in metabolic studies, since the formation of the hydroxylamine reactive metabolite cannot be demonstrated in vivo. For prochloraz, we observed that residue levels are significantly higher in males than in females for gill, fat, brain and carcasses, however, the reasons for this difference remain unclear. Although, the presence of glucuronide conjugates was detected in vivo and in vitro, the chemical structure of isolated metabolites has to be determined. However, the comparison of the in vivo versus in vitro metabolic profiles indicates that several peaks, probably corresponding to intermediate metabolites, were present only in hepatocyte incubations. Biotransformation of NP2EO occurred in vivo and in vitro in rainbow trout, but did not result in the formation of 4-n-NP. The major metabolite present in bile corresponded to the NP2EO-glucuronide but this metabolite was not found in vitro. It is concluded that hepatocytes may produce a different metabolic pattern than in the whole fish, but may also give evidence of a metabolic pathway difficult to apprehend in vivo.     

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis.

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO - Igepal(R) 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine (3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations > or =10 micromol/l. The presence of IGF-I (10-100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25-50 micromol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1-100 micromol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 micromol/l), NP2EO and NP (30 micromol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1-100 nmol/l 17beta-estradiol had no effect in our in vitro system, Triton(R) X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.     

Characterization and induction of xenobiotic metabolizing enzyme activities in a primary culture of rainbow trout hepatocytes

1. A primary cell culture from rainbow trout (Oncorhynchus mykiss) liver was prepared and evaluated for biotransformation of xenobiotics.

2. The hepatocytes maintained cytochrome P-450 content, as well as their cytochrome P-450-dependent activities, stable for 5–6 days in serum-free medium. Protein and glutathione levels, as well as other enzyme activities important for biotransformation, were close to their fresh cell levels throughout the culture period.

3. The cells were also responsive to cytochrome P-450 inducers. Both β-naphthoflavone (BNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused an increase in ethoxyresorufin O-deethylase (EROD) activity, which was dose-dependent over the concentration ranges of 3.6–360 nM and 2.5–100 pM, respectively. The induced activities in BNF-exposed cells returned to basal levels within 48 h after replacing the medium with a BNF-free medium. Exposure of cells to TCDD (100 pM) for 48 h induced EROD activity which, in contrast to response of BNF-exposed cells, continued to increase after the medium had been replaced with TCDD-free medium.

4. The results show that trout hepatocytes in primary culture afford a reliable in vitro method for studying the regulation and functions of xenobiotic biotransformation enzymes, and for defining toxic effects of aquatic pollutants in cells.     

Characterization and induction of xenobiotic metabolizing enzyme activities in a primary culture of rainbow trout hepatocytes

1. A primary cell culture from rainbow trout (Oncorhynchus mykiss) liver was prepared and evaluated for biotransformation of xenobiotics. 2. The hepatocytes maintained cytochrome P-450 content, as well as their cytochrome P-450-dependent activities, stable for 5-6 days in serum-free medium. Protein and glutathione levels, as well as other enzyme activities important for biotransformation, were close to their fresh cell levels throughout the culture period. 3. The cells were also responsive to cytochrome P-450 inducers. Both beta-naphthoflavone (BNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused an increase in ethoxyresorufin O-deethylase (EROD) activity, which was dose-dependent over the concentration ranges of 3.6-360 nM and 2.5-100 pM, respectively. The induced activities in BNF exposed cells returned to basal levels within 48 h after replacing the medium with a BNF-free medium. Exposure of cells to TCDD (100 pM) for 48 h induced EROD activity which, in contrast to response of BNF-exposed cells, continued to increase after the medium had been replaced with TCDD-free medium. 4. The results show that trout hepatocytes in primary culture afford a reliable in vitro method for studying the regulation and functions of xenobiotic biotransformation enzymes, and for defining toxic effects of aquatic pollutants in cells.     

Effect of the fungicide benomyl on xenobiotic metabolism in rats.

The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h following the treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route-independent effect on MFOs and is not toxic to the liver.     

The metabolism and excretion of prochloraz,an imidazole-based fungicide,in the rat

1. Following oral administration of prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy)ethylcarbamoyl]imidazole) at 100mg/kg body weight to rats, the compound underwent extensive metabolism, the primary route appearing to be opening of the imidazole ring followed by hydrolysis of the alkyl chain. The major metabolites were 2,4,6-trichlorophenoxyacetic acid and 2-(2,4,6-trichlorophenoxy)ethanol, which is present mainly as a glucuronide conjugate. Ring hydroxylation occurred to produce several minor metabolites. No unchanged prochloraz was excreted in the urine.

2. Tissue residues 96?h after dosing were generally < 1?mg prochloraz equivalents/kg tissue. The highest residues were found in the liver (2˙8–5˙1?mg prochloraz equivalents/kg tissue) and kidney (1˙5–2˙1?mg prochloraz equivalents/kg tissue), the principal organs of metabolism and excretion. Residues in female rats were generally slightly higher than those found in males.

3. The metabolites were quantitatively excreted within 96?h, with > 50% of the dosed radioactivity being found in the 0–24?h excreta. Urinary excretion accounted for 65% dose in male and 41% in female rats, respectively.     

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies

1.?In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000?g supernatants (S9) in environmental risk assessment.

2.?The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%).

3.?The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent).

4.?Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions.

5.?Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥1?h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.     

In vivo and in vitro effects of amrinone and milrinone on hepatic xenobiotic metabolism in rats

Studies were performed on the response of hepatic xenobiotic metabolizing enzymes to in vitro and in vivo exposure to amrinone and milrinone, two new inotropic compounds used in congestive heart failure. Both drugs exerted selective effects on various cytochrome P-450-dependent metabolic activities as well as conjugating pathways. Aminopyrine N-demethylation was selectively inhibited by in vitro addition of milrinone but not amrinone, and laurate hydroxylation was inhibited by both drugs. Cytosolic glutathione-S-transferase activity was profoundly inhibited by in vitro addition of both drugs. In vivo administration of either drug did not lead to significant inhibition of the pathways studied other than laurate hydroxylation which was depressed 20-30%. Irreversible binding of [14C]-amrinone-derived radioactivity to microsomal protein was partially NADPH-dependent. Inhibition by SKF 525-A, alpha-naphthoflavone and various antioxidants was observed. No binding of [14C]-milrinone-derived radioactivity was seen. It is suggested that amrinone may selectively inhibit certain hepatic drug-metabolizing enzymes through metabolic electrophilic intermediates.     

The metabolism and excretion of prochloraz, an imidazole-based fungicide, in the rat.

1. Following oral administration of prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy)ethylcarbamoyl]imidazole) at 100 mg/kg body weight to rats, the compound underwent extensive metabolism, the primary route appearing to be opening of the imidazole ring followed by hydrolysis of the alkyl chain. The major metabolites were 2,4,6-trichlorophenoxyacetic acid and 2-(2,4,6-trichlorophenoxy)ethanol, which is present mainly as a glucuronide conjugate. Ring hydroxylation occurred to produce several minor metabolites. No unchanged prochloraz was excreted in the urine. 2. Tissue residues 96 h after dosing were generally less than 1 mg prochloraz equivalents/kg tissue. The highest residues were found in the liver (2.8-5.1 mg prochloraz equivalents/kg tissue) and kidney (1.5-2.1 mg prochloraz equivalents/kg tissue), the principal organs of metabolism and excretion. Residues in female rats were generally slightly higher than those found in males. 3. The metabolites were quantitatively excreted within 96 h, with greater than 50% of the dosed radioactivity being found in the 0-24 h excreta. Urinary excretion accounted for 65% dose in male and 41% in female rats, respectively.     

Effects of steroid hormones in vitro on adrenal xenobiotic metabolism in the guinea pig

Studies were carried out to determine the effects of steroid hormones in vitro on adrenal and hepatic microsomal benzphetamine demethylation and benzo[a] pyrene hydroxylation. Testosterone inhibited adrenal drug metabolism but had no effect on hepatic enzymes, whereas 6β-hydroxytestosterone had no effect in either tissue. All of the corticosteroids tested (cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone) produced a concentration-dependent inhibition of adrenal drug metabolism, but had little or no effect on hepatic metabolism. The 17-deoxy-steroids were more potent inhibitors of adrenal metabolism than were their 17-hydroxylated counterparts. Cortisol was a potent inhibitor of adrenal benzphetamine and benzo[a]pyrene metabolism, produced a type I difference spectrum in adrenal microsomes, and diminished the magnitude of the benzphetamine-induced spectrum; 6 β-hydroxycortisol had none of these effects. Prior addition of benzphetamine to adrenal microsomes reduced the size of cortisol-induced spectral change. The results demonstrate that the effects of corticosteroids in vitro are relatively specific for adrenal enzymes and established a close association between the 6 β-hydroxylase and some drug-metabolizing enzymes. Adrenal steroids may have an important role in the regulation of adrenal xenobiotic metabolism.     

Elimination and metabolism of permethrin isomers in rainbow trout

Although permethrin, a synthetic pyrethroid insecticide, is highly toxic to fish, its toxicity to mammals is comparatively low. The distribution and metabolism of the cis- and trans-permethrin isomers were studied in rainbow trout to evaluate the role of these parameters in the differential toxicity of permethrin to fish and mammals. Both [¹⁴C]permethrin geometrical isomers were readily taken up and eliminated by rainbow trout. Elimination half-lives for [¹⁴C]permethrin residues in trout tissues, with the exception of fat, were in the magnitude of hours. High concentrations of a polar metabolite were found in bile within 4 hr of cis- and trans-permethrin exposure. Analysis by β-glucuronidase treatment, analytical thin-layer chromatography, and gas chromatography-mass spectroscopy indicated that the metabolite was the glucuronide conjugate of 4′-HO-permethrin. Urine contained a small amount of a polar metabolite that was resistant to hydrolysis by β-glucuronidase but was cleaved to some extent by aryl sulfatase. The relative absence of permethrin hydrolysis products in trout bile and the small amount of radioactivity excreted in urine suggested that the ability of rainbow trout to hydrolyze permethrin, in vivo, was minimal.     

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss)

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 μg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.     

Effects of dissolved metals and other hydrominerals on in vivo intestinal zinc uptake in freshwater rainbow trout

For aquatic organisms, zinc is both an essential nutrient and an environmental contaminant. The intestine is potentially the most important route of zinc absorption, yet little is known regarding this uptake pathway for zinc in fish. A recently developed in vivo perfusion system was used to investigate the effect of luminal composition upon intestinal zinc uptake in freshwater rainbow trout (Oncorhynchus mykiss). Perfusate cadmium and copper had specific, yet distinct, antagonistic effects upon lumen to tissue zinc movement. Copper significantly reduced the proportion of zinc taken up from the perfusate, and concomitantly limited the passage of zinc into the circulation and beyond. Conversely, cadmium decreased subepithelial zinc accumulation, with rates falling to 29 nmol g(-1) h(-1) from the control (zinc alone) values of 53 nmol g(-1) h(-1). Calcium had a similar action to copper, also reducing post-intestinal zinc accumulation from 0.06 to 0.02 nmol g(-1) h(-1), an effect attributed to interactions between calcium and the zinc uptake pathway. In addition to these effects, luminal composition also had a marked influence upon epithelial response to zinc. Calcium, copper and magnesium all greatly reduced zinc-induced mucus secretion. Cadmium, a toxic metal, significantly increased mucus secretion. It is proposed that these modifications were related to the essentiality of each element, and their potential mechanisms of uptake. Despite changes at the epithelium, the post-epithelial accumulation of zinc was dependent mainly upon the nature of the competing cation. Intestinal saline ion substitution experiments suggested a potential link of potassium ion efflux to zinc uptake. The effect of pH buffering of luminal solutions was also investigated.     

Effects of metronidazole on midazolam metabolism in vitro and in vivo

OBJECTIVE: Case reports have described elevated concentrations of CYP3A4 substrates (e.g. cyclosporin) during metronidazole treatment. Therefore, we wanted to study whether metronidazole affects CYP3A4 activity, using midazolam as a model substrate in vitro and in vivo. METHODS: In the in vitro part of the study, the effects of various concentrations of metronidazole (0-500 microM) on the formation of 1'-hydroxymidazolam from midazolam were studied using human liver microsomal preparations (n = 4). In the in vivo part, the effects of metronidazole on the pharmacokinetics and pharmacodynamics of oral midazolam were evaluated in a randomised, placebo-controlled cross-over study in ten healthy subjects. The subjects took either 400 mg metronidazole or matched placebo orally twice daily for 3 days. On day 3, 15 mg midazolam was administered orally. Plasma concentrations of midazolam and 1'-hydroxymidazolam were determined up to 24 h. The effects of midazolam were measured up to 10 h. RESULTS: Metronidazole (10-500 microM) showed no inhibitory effect on 1'-hydroxymidazolam formation by human liver microsomes. In healthy volunteers, metronidazole had no statistically significant effects on the pharmacokinetics of midazolam and 1'-hydroxymidazolam, and also the ratio of 1'-hydroxymidazolam to midazolam in plasma remained unchanged by metronidazole. The four employed psychomotor tests did not show significant differences between the metronidazole and placebo phases. CONCLUSION: Metronidazole had no effects on the 1'-hydroxylation of midazolam in vitro or on the pharmacokinetics and pharmacodynamics of midazolam in vivo. These findings indicate that metronidazole is not an inhibitor of CYP3A4.     

Effects of alkylphenols on bone metabolism in vivo and in vitro

Alkylphenols are endocrine disruptors that show estrogen-like effects in various wildlife species. However, little information is available about the action of these chemicals on bone metabolism. We investigated the effects of alkylphenols, such as nonylphenol (NP) and octylphenol (OP), on the formation of bone using several culture systems for osteoclasts and osteoblasts, as well as in vivo experiments. NP and OP dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) in cocultures of mouse spleen cells or mouse bone marrow cells with ST2 cells. However, beta-estradiol at 10(-9)M to 10(-6)M did not affect this process. In contrast, neither compound affected the proliferation and differentiation of rat calvarial osteoblast-like cells (ROB cells). When NP or OP (0.1mg/kg body weight) was administered subcutaneously to pregnant mice at 10 days, 12 days and 14 days post-coitus, fetuses at 17.5 days post-coitus showed stimulation of sternebrae bone calcification. Our findings suggest that alkylphenols have critical effects on the formation of bone by non-estrogenic effects.     

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse.

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24h. The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity. Conversely, pre-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action.     

Estrogenic effects in vitro and in vivo of the fungicide fenarimol

The fungicide fenarimol has the potential to induce endocrine disrupting effects via several mechanisms since it possesses both estrogenic and antiandrogenic activity and inhibits aromatase activity in cell culture studies. Hence, the integrated response of fenarimol in vivo is not easy to predict. In this study, we demonstrate that fenarimol is also estrogenic in vivo, causing significantly increased uterine weight in ovariectomized female rats. In addition, mRNA levels of the estrogen responsive gene lactoferrin (LF) were decreased in uteri, serum FSH levels were increased, and T3 levels decreased in fenarimol-treated animals. To our knowledge, only two other pesticides (o,p-DDT and methoxychlor) have previously been reported to induce an estrogenic response in the rodent uterotrophic bioassay. A pronounced xenoestrogenicity in serum samples from rats treated with fenarimol and estradiol benzoate (E2B) separately or in combination was observed, demonstrating the usefulness of this approach for estimating the integrated internal exposure to xenoestrogens. The MCF-7 cell proliferation assay was used to investigate further the dose-response curves for the estrogenic, antiestrogenic, and aromatase inhibiting properties of fenarimol in vitro. The results indicates that fenarimol exhibits a dual effect being aromatase inhibitor at low concentrations and estrogenic at higher concentrations.