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Archives of Toxicology (v.76, #12)

Effects of combined exposure to lead and cadmium on pituitary membrane of female rats by Anil Pillai; P.N. Laxmi Priya; Sarita Gupta (pp. 671-675).

The effects of combined exposure to lead and cadmium on pituitary membrane were studied. Adult female rats were treated intraperitoneally with either lead acetate and cadmium acetate alone or in combination at a dose of 0.05 mg/kg daily for 15 days. Both metals accumulated in the pituitary after the exposure. The membrane fluidity was decreased after the heavy-metal treatment. Among the three groups cadmium treatment showed more effect than did other treatments and the combined treatment showed intermediate values. Na⁺K⁺ATPase activity was decreased significantly by cadmium and combined treatments. The Schiff's base and inorganic peroxide levels were increased after the metal exposure. In conclusion, exposure to lead and cadmium caused accumulation of the metals in the pituitary and lowered the membrane fluidity, which may affect membrane function and cause alterations in receptor binding and secretory mechanism(s) of pituitary hormones. The combined treatment with metals produced intermediate results.

Keywords: Cadmium Fluidity Lead Na+K+ATPase

Metabolism and excretion of 2-nitro-p-cresol in rats by Hideo Kurebayashi; Seiichi Nambaru; Masamichi Fukuoka; Tsutomu Yamaha; Akira Tanaka (pp. 676-681).

Metabolism of 2-nitro-p-cresol (NPC), an important commercial chemical, was studied in female Sprague-Dawley rats, using ¹⁴C-NPC. It was found that NPC was rapidly absorbed and excreted after an oral dose of 250 mg/kg. Approximately 90% of the administered dose was excreted into urine and less than 10% of the dose into feces for 5 days. Urinary and fecal excretion were found to the same extent after 48 h. Bile excretion amounted to approximately 25% for 2 days. Blood levels of ¹⁴C-NPC reached the maximum concentration (39.4 µg-equivalents/g) within 1 h, and decreased bi-exponentially. The apparent half-lives of ¹⁴C-NPC were 3.8 h for the rapid phase and 37 h for the slow phase, respectively. From studying the distribution in organs at 1.5, 6, 24, 72 and 120 h, we found that the concentrations of radioactivity in various tissues of rats were relatively high in the stomach, intestine, liver, kidney, blood, ovary and uterus. Most organs showed the maximum concentrations at 1.5 h, except for intestine, kidney, ovary, and uterus at 6 h. There was no specific tissue retention after 72 h. Two main conjugate metabolites, glucuronide and sulfate of NPC, were detected with free NPC and 2-acetylamino-p-cresol (AAPC) in the urine. NPC was rapidly absorbed and excreted mainly into urine as the conjugate metabolites. A part of NPC was reduced to 2-amino-p-cresol, followed by acetylation to give AAPC.

Keywords: 2-Nitro-p-cresol Female rat Absorption Organ distribution Excretion Metabolism Glucuronide Sulfate 2-Acetylamino-p-cresol

Comparative toxicokinetic/toxicodynamic study of rubber antioxidants, 2-mercaptobenzimidazole and its methyl substituted derivatives, by repeated oral administration in rats by Kazue Sakemi; Rieno Ito; Takashi Umemura; Yasuo Ohno; Mitsuhiro Tsuda (pp. 682-691).

2-Mercaptobenzimidazole (MBI), a rubber antioxidant, is known to exhibit potent thyroid toxicity in rats, whereas its methylated derivatives are much less toxic. To characterize this methyl-substituent effect on the thyroid toxicity of MBI, comparative toxicokinetic analyses have been conducted in the present study. MBI and the MMBIs [4-methylated MBI (4-MMBI) and 5-methylated MBI (5-MMBI), and a 1:1 mixture of these 4- and 5-methylated isomers (MMBI mix)] suspended in corn oil were repeatedly administered (at 0.3–0.6 mmol/kg) to male Wistar rats by gavage once daily for 2 weeks. After the first and last administrations, blood and urine samples were collected, and the levels of unchanged compounds and their desulfurated metabolites were determined by high performance liquid chromatography. After repeated oral administration (roa), the C_max and area under concentration-time curve (AUC) of MBI were markedly increased, while the MMBIs essentially were cleared from the blood within 10 h. After roa, the C_max and AUC of 4-MMBI decreased markedly, suggesting metabolic enzyme induction. However, the toxicokinetic parameters of 5-MMBI were not markedly altered by roa. The inhibitory potencies (IC₅₀) against lactoperoxidase of MBI, 4-MMBI, and 5-MMBI were 20.6 µM, 45.6 µM and 31.6 µM, respectively. Thus, we suggest that the marked decrease of thyroid toxicity by methyl substitution of MBI is caused mainly by a decrease in systemic exposure to the compounds and partly by a decrease in inhibition of thyroid hormone synthesis.

Keywords: Thyroid toxicity Thioureylenes Toxicokinetics/toxicodynamics Repeated oral administration Rats 2-Mercaptobenzimidazole 2-Mercapto-4-methylbenzimidazole 2-Mercapto-5-methylbenzimidazole

Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor by C. Latchoumycandane; P. Mathur (pp. 692-698).

Methoxychlor is one of the environmental contaminants that has been shown to induce reproductive abnormalities in male rats. The mechanism of action of methoxychlor on the male reproductive system remains unclear. In the present study we have sought to investigate whether short-term administration of methoxychlor induces oxidative stress in the testis of adult rats. Methoxychlor (50, 100, or 200 mg/kg body weight per day) was administered orally for 1, 4, or 7 days. The animals were killed using anesthetic ether on the day following the last dosing. The weights of epididymides, seminal vesicles, and ventral prostate decreased after 50, 100, or 200 mg/kg per day for 7 days but remained unchanged after 1 and 4 days of treatment. The production of superoxide anion and hydrogen peroxide increased in the animals that received methoxychlor for 4 and 7 days. The activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased, while the level of lipid peroxidation increased in the testis after 4 or 7 days of treatment. The results indicated that short-term exposure to methoxychlor induces oxidative stress in the testis by decreasing antioxidant enzymes and increasing lipid peroxidation, possibly by inducing reactive oxygen species. In conclusion, the adverse effect of methoxychlor on the male reproduction could be due to induction of oxidative stress in testis.

Keywords: Methoxychlor Testis Superoxide anion Antioxidant enzymes Lipid peroxidation Daily sperm production Rat

Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis by Wolfram Parzefall; Eveline Kainzbauer; Hong-Min Qin; Monika Chabicovsky; Rolf Schulte-Hermann (pp. 699-706).

The mouse strain C3H shows high incidence of liver tumors in carcinogenicity testing, while the strain C57BL exhibits low incidence. The F1 generation hybrids, B6C3F1, which are widely used in long-term carcinogenesis bioassays, are of intermediate sensitivity. We asked whether this strain difference could be due to different susceptibility of the parenchymal cells to signals inducing replication or apoptosis. Hepatocytes were isolated and cultured according to standard protocols. We tested (1) for the induction of DNA synthesis by epidermal growth factor (EGF), (2) for its inhibition by TGF-β1, and (3) for the induction of apoptosis by TGF-β1. Basal rates of DNA synthesis in untreated hepatocytes cultured from C3H and B6C3F1 mice were 6.5 and 3.5 times higher, respectively, than in hepatocytes from C57BL on day 3. Moreover, addition of EGF (10 ng/ml) increased DNA synthesis on day 3 in hepatocytes from C3H (4.2-fold) and B6C3F1 (2.7-fold) more strongly than in hepatocytes from C57BL. Treatment with TGF-β1 inhibited basal and EGF-stimulated DNA synthesis dose-dependently. Inhibition was maximal at 1 ng TGF-β1/ml in cultures from C57BL mice, and at 0.3 ng/ml in hepatocytes from C3H mice. In untreated hepatocytes from both strains virtually no apoptotic figures (condensed or fragmented nuclei, Hoechst 33285 staining) were found. After treatment with TGF-β1 the incidence of apoptotic nuclei in hepatocytes from C57BL was higher than in cells from C3H mice (1.7% vs 3% on day 3). Thus it appears that hepatocytes from C57BL mice possess a lower growth potential, as indicated by a low basal rate of DNA synthesis and low inducibility by EGF, but a higher sensitivity to induction of apoptosis by TGF-β1 than hepatocytes of the C3H strain. These findings may be helpful to explain the different susceptibility to induction of hepatocarcinogenesis in C3H and C57BL mice.

Keywords: Mouse Rat Carcinogenesis Short-term culture DNA synthesis Apoptosis Transforming growth factor β-1 Epidermal growth factor

Potentiation of 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123)-induced liver toxicity by ethanol in guinea-pigs by Perrine Hoet; Jean-Pierre Buchet; Christine Sempoux; Vincent Haufroid; Jacques Rahier; Dominique Lison (pp. 707-714).

HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane), a substitute for the banned chlorofluorocarbons (CFCs), is a structural analogue of the well-known hepatotoxicant halothane. The objectives of these experiments were to investigate (1) whether, like halothane, multiple exposure increases the risk of HCFC-123-induced liver toxicity, and (2) whether ethanol, a potent CYP2E1 inducer, potentiates the liver toxicity of HCFC-123. In experiment 1, male Hartley guinea-pigs were exposed twice a week to 5000 ppm HCFC-123 (4 h) during 3 weeks followed by 2 weeks recovery, and then re-exposed or not during 4 h to 5000 ppm HCFC-123. A group with a single exposure to 5000 ppm HCFC-123 and a control group were also included. In experiment 2, guinea-pigs received 5 or 10% ethanol in drinking water during 12 days before a single 4-h exposure to 5000 ppm HCFC-123. A group receiving 10% only, a group exposed once to 5000 ppm HCFC-123 but not pre-treated with ethanol and a control group were also included. In both experiments, the liver toxicity was assessed, 24 h post-exposure, by the serum activities of alanine aminotransferase (ALT) and isocitrate dehydrogenase (ICDH) as well as by histopathology. In experiment 2 the urinary excretion rate of the main metabolites trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA) was assessed and CYP2E1 activity was measured by the chlorzoxazone metabolic ratio. Multiple exposure to 5000 ppm HCFC-123 did not cause greater liver damage than a single exposure (ALT, ICDH 3-fold control values). At this level of exposure the liver lesions were totally reversible within two weeks. Ethanol consumption produced CYP2E1 induction, increased urinary excretion of both HCFC-123 metabolites (more than 2-fold the rate measured in the non-induced group) and markedly increased the liver toxicity of HCFC-123 as shown by the serum liver enzyme activities (ALT 8.5-fold increase, ICDH 13-fold increase), and the histopathology. The necrosis was predominantly localised in the intermediate zone of the hepatic lobules with vacuolisation of the centrilobular zones. The effects associated with 10% ethanol pre-treatment were less marked than those observed with ethanol 5% and could be explained by the remaining blood ethanol levels causing an inhibition of HCFC-123 biotransformation. Significant correlations were obtained between the serum enzyme activities, the histopathology, the excretion rate of the metabolites and CYP2E1 activity. It can be concluded that (1) multiple exposure to HCFC-123 did not increase the liver toxicity of HCFC-123 in this experimental model, and (2) chronic ethanol consumption, known to be CYP2E1 inducer, strongly enhanced the biotransformation of HCFC-123 and its liver toxicity.

Keywords: HCFC-123 CYP2E1 induction Hepatotoxicity Guinea-pigs

Involvement of nitric oxide in myotoxicity produced by diisopropylphosphorofluoridate (DFP)-induced muscle hyperactivity by Ramesh C. Gupta; Dejan Milatovic; Wolf-D. Dettbarn (pp. 715-726).

Oxidative stress, as determined by increased lipid peroxidation, has been implicated in the pathology of myotoxicity. As a model system to study the response of muscle to oxidative insults, we have studied the effects of diisopropylphosphorofluoridate (DFP)-induced muscle hyperactivity on levels of nitric oxide (NO) and energy metabolites in rat skeletal muscles. In in vivo experiments, citrulline levels as indicators of NO and NO synthase (NOS), and ATP and phosphocreatine (PCr) as indicators of mitochondrial dysfunction, were determined using HPLC methods 15 min, 30 min, 60 min, 2 h, and 24 h after intoxication. Within 15 min of DFP exposure, with onset of fasciculations, citrulline levels were significantly elevated in all three muscles [soleus, extensor digitorum longus (EDL), and diaphragm]. Maximum increases in citrulline (272–288%) were noted 60 min after DFP injection. At this time point, acetylcholinesterase activity was reduced by 90–96% (soleus < diaphragm < EDL). The levels of ATP and PCr were maximally reduced (30–43%), and total adenine nucleotides, and total creatine compounds showed declines. The findings revealed that the increase in NOS activity and NO was greater than the decrease of ATP and PCr. Since memantine (MEM) has been shown to reduce nerve and muscle hyperactivity, we have studied the possible protective effect of MEM on the DFP-induced biochemical changes. Pretreatment with MEM (18 mg/kg s.c.) and atropine sulfate (16 mg/kg s.c.), 60 min and 15 min, respectively, before DFP injection prevented the increase in citrulline and muscle hyperactivity and the decrease in ATP and PCr. These data suggest that free radical reactions by depleting high-energy phosphates may be initiating the cascade of events leading to myotoxicity during DFP-induced muscle hyperactivity.

Keywords: Organophosphate Diisopropylphosphorofluoridate (DFP) Memantine Atropine Nitric oxide Citrulline ATP Energy metabolites Acetylcholinesterase Excitotoxicity Oxidative stress Myotoxicity

Benzophenone-induced estrogenic potency in ovariectomized rats by Yoshio Nakagawa; Kuniaki Tayama (pp. 727-731).

To assess the estrogenic potency of benzophenone by in-vivo uterotrophic assay, we gave orally either the compound (100 or 400 mg/kg) or 17β-estradiol (0.2 mg/kg) as a positive control, once per day for 3 days, to ovariectomized Sprague-Dawley (SD) rats, and all rats were killed 24 h after being given the last dose. The high dose of benzophenone elicited an approximately 1.9-fold increase in absolute and relative uterine weight, and 17β-estradiol increased uterine weight approximately fivefold relative to the control. The uterine response caused by both compounds was accompanied by an increase in luminal epithelium height and stromal cell numbers in the uterus and an increase in the thickness of vaginal epithelium cell layers with cornification. At 24 h after the last dose, the mean serum concentrations of benzophenone, benzhydrol and p-hydroxybenzophenone in the high-dosed rats were 10.4±1.0, 1.5±0.3, and 0.7±0.2 (mean ± SE) µmol/l, respectively, whereas in the serum of low-dosed rats these compounds were not detected. When a single oral administration of benzophenone (100 or 400 mg/kg) was given to intact female rats, serum concentrations of benzophenone, benzhydrol and p-hydroxybenzophenone increased in a dose-dependent manner 6 h later. Previously, Nakagawa et al. (2000) and Nakagawa and Tayama (2001) reported that the subcutaneous injection of p-hydroxybenzophenone into juvenile female rats elicited estrogenic activity in reproductive organs, whereas neither benzophenone nor benzhydrol had such an effect. In addition, p-hydroxybenzophenone itself rather than the parent compound caused a proliferation of estrogen receptor-positive MCF-7 cells in vitro. Based on these findings, it is apparent that the pro-estrogenic compound benzophenone requires biotransformation to p-hydroxybenzophenone, a metabolite with intrinsic hormonal activity.

Keywords: Benzophenone Oral administration Metabolites Uterotrophic assay Estrogenic activity

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Archives of Toxicology (v.76, #12)