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The carcinogenicity hazard of chemicals in humans has been determined primarily on the basis of long-term animal testing. As progress has been made in determining the modes of action (MOAs) of chemicals that produce neoplasia in animal assays, it has become clear that there are qualitative and quantitative species differences between laboratory animals and humans, and it has become increasingly important to evaluate the relevance of these MOAs to humans. In this review, we discuss the usefulness of a framework for assessment of MOAs of nongenotoxic and genotoxic carcinogens and its human relevance, and remaining challenges to quantitative assessment of human risk.

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

Although benomyl and its metabolite carbendazim have been shown to adversely affect male reproduction, the mechanisms of action do not appear to involve the endocrine system. However, few studies have been conducted using currently proposed tests specifically focused on endocrine disruption. Here, potential estrogen- and androgen-mediated activity of benomyl was therefore investigated in vitro and in vivo. Benomyl and carbendazim proved negative for agonistic and antagonistic activity in reporter gene assays for the human estrogen receptor alpha and androgen receptor. In uterotrophic and Hershberger assays using Crj:CD(SD)IGS rats, benomyl (100, 300 or 1000 mg/kg/day, p.o., N = 6) did not exert agonistic effects. However, the highest dose decreased uterine weights in the uterotrophic assay, and decreased weights of some androgen-related tissues of castrated rats receiving a testosterone propionate (TP, 0.2 mg/kg) injection in the Hershberger assay; the effects were less severe than those with p,p'-DDE (100 mg/kg/day). When 4 mg/kg/day of TP was injected, decrease of organ weights due to benomyl was attenuated but still observed. Thus, its influence in some tissues was more potent than that of p,p'-DDE. Benomyl had no apparent effects on serum androgen levels. Microarray analysis of the gene expression profile in the ventral prostate of TP-injected castrated rats treated with benomyl indicated clear differences from the patterns observed with p,p'-DDE and flutamide. Taken together, these findings suggest the decreased organ weights observed in vivo to be caused by mechanisms that are not steroid-receptor-mediated, such as interfering with assembly of microtubules by benomyl. The study furthermore suggests that functional genomics may provide a reliable evidence for accurate categorization of test chemicals.

Development of an internationally recognized standard for the Hershberger assay as a screening tool to detect potential (anti-)androgenic chemicals is in progress. In the present preliminary study, we evaluated the reliability of the enhanced Hershberger assay to detect thyroid hormone modulating activity, while concentrating attention on possible confounding influence on evaluation of (anti-)androgenic activity. Castrated or testosterone propionate (TP; 0.2 or 0.25 mg/kg/day)-injected castrated male Crj:CD(SD) IGS rats (seven weeks of age) were dosed for 10 days by oral gavage with vehicle (corn oil) or the following chemicals: propylthiouracil (PTU; 2.5 mg/kg/day), a potent inhibitor of thyroid hormone synthesis, phenobarbital (PB; 125 mg/kg/day) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE; 100 mg/kg/day), two hepatic enzyme inducers that enhance the clearance of thyroid hormones. PTU markedly increased thyroid weights, and decreased serum T3 and T4, and increased serum TSH, also causing marked microscopic alteration of the thyroid gland. In comparison, PB and p,p'-DDE only significantly affect serum T4 and revealed some histopathological findings. The alterations appeared to be more robust in the presence of TP. Furthermore, data for p,p'-DDE demonstrated its anti-androgenic effects, whereas PTU and PB had little or no effects on the weights of androgen-related accessory glands/tissues: the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, glans penis, Cowper's glands, and levator ani plus bulbocavernosus (LABC) muscles. Weight of the LABC muscles was decreased by PB treatment in TP-treated castrated rats. These findings in the present study suggests that the enhanced Hershberger assay, with evaluation of thyroid histopathology and weights, and hormone levels, appears to be reliable for screening for not only (anti-)androgenic chemicals but also thyroid hormone modulators. In order to evaluate whether the sensitivity and specificity of such a thyroid assay is great enough for routine screening purposes, future experiments including dose-response studies using lower dose levels have to be performed.

Pregnant rats were administered flutamide at a daily dose of 10 mg/kg from gestation day 12 (GD12) to GD21, or 30 mg/kg on 2 successive days in the period from GD14 to 21 (GD14-15, GD16-17, GD18-19, and GD20-21). The effects on ventral prostate in male offspring were examined by RT-PCR on postnatal day 1 (PND1) for the 10 mg/kg group, and by receptor binding assay at PND76 or 78 and morphologically at PND7, 14, and 21 for all groups. RT-PCR demonstrated increase in mRNAs for the androgen receptor (AR) and the keratinocyte growth factor (KGF), but not transforming growth factor (TGF)-beta1 and beta2, the epidermal growth factor receptor (EGFR), and the vascular endothelial growth factor (VEGF). Prostate tissue showed a consistent reduction in the number of main ducts and ductal branchpoints, as well as the complexity of the terminal ductal network in the 10 mg/kg group, and the 30 mg/kg GD16-17 and GD18-19 groups. The effect on ductal architecture was severest with the 10 mg/kg regimen. The organ weights at PND76 or 78 were reduced in all flutamide treated groups. The value for the 10 mg/kg group was lowest, while the 30 mg/kg GD14-15 and GD20-21 groups were least affected. Receptor binding assays exhibited no significantly difference regarding maximum binding capacity (Bmax) and dissociation constant (Kd) between the control and any flutamide treated group. In conclusion, prenatal exposure to flutamide caused increased AR and KGF mRNA expression just after exposure, and an irreversible morphological change that is severest when the prostatic buds are developing in the ventral prostate. However the receptor binding capacity later in life was not affected.

In the present study, we investigated epithelial cell proliferation in heterozygous p53 knockout (p53+/-) mice after administration of two urinary bladder non-genotoxic carcinogens, in comparison with that in wild-type littermates (p53+/+). Mice at 10-weeks of age were given 2% sodium ortho-phenylphenate (Na-OPP) or 2.5% uracil in the basal diet for 4 weeks. Uracil evoked a marked elevation of epithelial cell proliferation and development of papillary or diffuse epithelial hyperplasia associated with calculus formation in p53+/- as well as p53+/+ mice. Administration of Na-OPP caused alkalization of the urine in both. Neither Na-OPP nor uracil induced a higher epithelial cell proliferative response in p53+/- mice as compared with p53+/+ mice. While we previously reported p53+/- mice to be highly sensitive to a genotoxic urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine, the present results suggest that p53+/- mice may not have a high susceptibility to induction of urinary bladder tumors by non-genotoxic carcinogens.

Flutamide, which has antiandrogenic properties, was administered to pregnant rats, and effects on male offspring were examined. Crj: CD (SD) IGS (SPF) females were administered flutamide (0.15, 0.6, 2.5, 10.0, 100 mg/kg, p.o.) from gestation Day 14 to post parturition Day 3. The number of pups, body weights, clinical features, anogenital distance (AGD), nipple retention, testicular descent, and urogenital malformation in F1 males were examined. Hormone measurement, necropsy and histopathological examination were carried out at post-neonatal Day 4 (PND 4) and PND 60. Sperm analysis was also carried out at PND 60. Decrease in body weight was seen in the 100 mg/kg group and the AGD was decreased at 2.5 mg/kg and above. Retention of nipples, hypospadia, vaginal pouches, penis malformation, unilateral ectopic testis, and decrease of organ weights (prostate, seminal vesicles, levator ani muscle plus bulbocavernosus muscle, testis) were observed at 10 mg/kg and above. Testicular testosterone (T) was increased significantly with 100 mg/kg at PND 4 and tendencies for increase were observed in serum T, LH and FSH at 10 mg/kg and more at the same time point. In contrast, elevated levels of LH and FSH were seen with 100 mg/kg at PND 60. Histopathological examination revealed defects or hypoplastic changes of genital organs (or = 10 mg/kg), squamous metaplasia (10 mg/kg) or mucification (100 mg/kg) of the urethral diverticulum epithelium and inflammation of genital organs (100 mg/kg). Though only undescended testes lacked spermatogenesis at 10 mg/kg, atrophic change of seminiferous tubules and azoospermia were observed in the 100 mg/kg group, despite testicular descent. Perinatal administration of flutamide affected F1 male rats at 2.5 mg/kg and above. In addition to urogenital malformation, 100 mg/kg flutamide caused high LH and FSH levels at PND 60. This study indicates that the most sensitive parameter is AGD, whereby reduction was observed at 2.5 mg/kg. A clear no-effect level (NOEL: 0.6 mg/kg) was obtained in this perinatal study of an antiandrogenic chemical.

Synthetic pyrethroids are among the most common pesticides and insecticides currently in worldwide use. Eriksson and co-workers postulated that oral exposure of mice to pyrethroids during a neonatal brain growth spurt induces permanent disturbance in the cerebral muscarinic cholinergic receptor (MAChR) and behaviour. However, the scientific basis for these phenomena is now under discussion. The present study was performed to determine whether the experimental findings of Eriksson's study could be reproduced in newborn mice by inhalation. Male and female NMRI mice were exposed to d-allethrin by whole-body inhalation for 6 h per day between postnatal days 10 and 16. Actual concentrations of d-allethrin were 0.43, 1.35, 3.49 and 74.2 mg m(-3) (equivalent to 0.70, 2.2, 5.7 and 120.2 mg kg(-1) day(-1), respectively), and the mass median aerodynamic diameter and geometric log-standard deviation of mist particles ranged from 2.58 to 2.98 micro m and from 1.58 to 2.09 micro m for all groups, respectively. The highest exposure level in the present study (74.2 mg m(-3)) was ca. 13,000 times as high as the concentration used in practice. The MAChR in the three brain areas (cortex, hippocampus and striatum) and motor activity were examined at the ages of 17 days and 4 months. In addition, a water-maze test was performed at the age of 11 months. There was no systemic toxicity interfering with the interpretation of assay results. The neonatal exposure to d-allethrin by inhalation did not induce effects either on the brain MAChR density and motor activity at 17 days and 4 months or on performance in the learning/memory test at the age of 11 months. The effects of allethrins on developmental neurotoxicity that Eriksson and co-workers reported previously were not reproduced in the present study.

In order to standardize test procedures for evaluation of neurotoxicity of chemical exposure, a collaborative study with a common protocol based on the Organization for Economic Cooperation and Development (OECD) test guideline TG407 was conducted by eleven laboratories, using acrylamide and 3,3’-iminodipropionitrile (IDPN) as positive neurotoxicants. This report summarizes the results of this neuropathological evaluation of neurotoxicity, when acrylamide (3, 10, and 30 mg/kg) and IDPN (20, 50, and 100 mg/kg) were orally administered to rats for 28 days. The study revealed typical alterations, including degeneration of peripheral nerve fibers and Purkinje's cell necrosis with acrylamide, and axonal swelling in the brainstem and spinal cord with IDPN. In addition to the neurotoxicity, IDPN exerted toxic effects on the cerebral arteries, hyalinization of vessel walls being observed. In general, the neuropathological findings well demonstrated the neurotoxicity of both chemicals. These results indicate that neuropathological evaluation can play a crucial role in screening for potential neurotoxicants with risk for human health, the expected neurotoxic effects being detected in this collaborative study consistently in all the laboratories.

A 5-day Hershberger assay, a 20-day pubertal male assay, a one-month oral administration study and a reproductive toxicity study were carried out using an antiandrogenic chemical, flutamide (FL). The effects of FL were as follows: weight decrease in male reproductive organs such as the prostate and seminal vesicles were observed at ≥0.6 mg/kg/day as the most sensitive parameter in the Hershberger assay and the one-month oral administration study. Reduction in number of spermatogenic cells was found at ≥0.6 mg/kg/day as the most sensitive parameter in the 20-day pubertal male assay. A decrease in the anogenital distance was observed at ≥2.5 mg/kg/day as the most sensitive parameter in the reproductive toxicity study. These effects were compared with the reported results of one-month oral administration study of an estrogenic chemical, ethinylestradiol (EE), and an androgenic chemical, methyltestosterone (MT). Differences in the results were as follows: serum hormone levels of LH and testosterone were increased by the FL-treatment, but decreased with EE despite testicular atrophy in all cases. While EE and MT produced atrophic changes in Leydig cells, FL induced their hyperplasia. Thus, different results were obtained regarding serum hormone levels and histopathological changes in Leydig cells with the endocrine disruptor. Attention should be concentrated on these two parameters to clarify and characterize the mechanisms of action of so called endocrine disruptors.

A 5-day Hershberger assay using young mature male rats to detect compounds interfering with androgen receptor (AR)-mediated mechanisms was evaluated for ability to identify p,p'-DDE (a weak AR antagonist) and methyltestosterone (MT, an AR agonist). Fenitrothion, an organophosphate pesticide, was also evaluated in this validated assay. Castrated male Crj:CD(SD)IGS rats (1 week after castration, 11 weeks of age) were subjected to experiments. To determine a suitable value of testosterone propionate (TP) as a reference androgen for detection of antiandrogenic chemicals, castrated male rats were treated daily with TP (0, 0.06, 0.25, 1, 4, or 16 mg/kg/day, s.c.). TP produced increases in weights of ventral prostate, seminal vesicles and levator ani plus bulbocavernosus muscles. Serum androgen level measured by RIA kit (mostly TP) were elevated in a dose-related manner, while the weights of organs with 1 mg/kg/day of TP were nearly equivalent to the maximum responses (i.e., sub-maximal). One hundred mg/kg/day of p,p'-DDE significantly attenuated TP 0.1 mg/kg-induced increases in weights of seminal vesicles and muscles, and TP 1 mg/kg-induced increases in weights of ventral prostate, seminal vesicles and muscles, but did not affect the weight of these organs in either TP 16 mg/kg-treated or intact rats, demonstrating that the dose range of 0.1-1 mg/kg TP is suitable for reference androgen. Oral treatment with 100 mg/kg of MT increased the weights of ventral prostate, seminal vesicles and muscles as strongly as did subcutaneous injection of 1 mg/kg of TP. These findings demonstrate that the 5-day Hershberger assay using young mature as well as immature male rats is a sensitive and valid short-term screening method for the detection of chemicals interfering with AR-mediated mechanisms. To determine whether fenitrothion interferes with AR-mediated mechanisms in vivo, fenitrothion (0, 0.75, 1.5 or 3 mg/kg/day) was administered by gavage for 5 days to castrated rats for androgenicity, or to castrated rats treated with 1 mg/kg TP for antiandrogenicity. Treatment with fenitrothion had no adverse effects on clinical signs, body weight, or liver or kidney weights, but cholinesterase activities in the brain and erythrocytes were significantly suppressed by fenitrothion to, respectively, 77-81% and 66-67% of control levels. In the antiandrogenicity experiment, serum androgen levels of TP-treated, castrated rats did not differ among groups. Treatment with 100 mg/kg of p,p'-DDE as a positive control again significantly attenuated TP-induced increases in weights of the ventral prostate and seminal vesicles, while fenitrothion had no effect on the weights of any organs. In the androgenicity experiment, treatment with 100 mg/kg of MT significantly increased weights of ventral prostate, seminal vesicles and muscles, but fenitrothion had no effects on the weights of any of these organs. These findings yield no evidence that fenitrothion interferes with AR-mediated mechanisms in vivo, consistent with the result of several toxicological bioassays.

2, 2, 4-Trimethylpentane (TMP) was administered orally to male Sprague-Dawley rats for 26 weeks to induce α2u-globulin nephropathy and histopathological examinations of the kidney as well as urinary sediment morphology and 5-bromo-2'-deoxyuridine (BrdU) incorporation were performed. The number of exfoliated renal epithelial cells in the urine increased with the TMP treatment until week 4, when a peak of approximately 90-fold as compared with the matched control values was observed. Significantly elevated levels then persisted throughout the study. Deposits of α2u-globulin and BrdU labeling in the kidney also reached maxima at week 4. A correlation among the amount of α2u-globulin deposits, the BrdU labeling indices, and the numbers of exfoliated cells in the urine was noted at each time point. The results thus suggest that damaged cells become detached following excessive accumulation of α2u-globulin and this stimulates regenerative cell proliferation in the kidney. Quantitative examination of exfoliated cells in the urine may thus provide a reliable estimate of cell proliferation induced by α2u-globulin nephropathy during rat toxicology studies.

The testicular toxicity of cyclophosphamide (CP) in rats and recovery of normal spermatogenesis over an 8-week period were assessed. Male rats were administered 40mg/kg CP by gavage daily for 1 week and sacrificed at 1 day, 3 weeks, and 8 weeks after the cessation of treatment. The numbers of seminiferous epithelial cells were counted in the seminiferous tubules in stages II-III, V, VII, and XII of the spermatogenic cycle. Spermatogonia were decreased in number at 1 day after the last treatment, but most of the surviving spermatogonia including type A had proliferative activity, as demonstrated by PCNA staining. After 3-week recovery, the numbers of spermatogonia were improved, but the numbers of spermatocytes and round spermatids were reduced in some stages. Furthermore, focal substantial changes such as atrophy of seminiferous tubules were present in the testis. After 8-week recovery, the numbers of spermatogenic cells up to round spermatids were comparable to those in control specimens, and histopathologically no remarkable changes were observed in the testis. However, lower weights and sperm counts for testis and epididymis were still found at this time. These findings suggested that histopathological changes as subtle as decreased number of spermatogonia should be detected in evaluations of male chemical reproductive toxicity. Our findings also suggested that PCNA staining may be useful for predicting reversibility of testicular toxicity.

A study was conducted to determine the mode of action for promoting effect of large amounts of vitamin A (VA) on cell proliferation of thyroid proliferative lesions induced by thiourea (TU) (Mitsumori et at, Cancer Letter 103: 19-31, 1996). Groups of male F344 rats (16/group) received 0.1% TU in water (TU group), 0.1% VA in diet (VA group), both 0.1% TU in water and 0.1% VA in diet (TU+VA group) or distilled water and basal diet (control group) for 4 weeks. Increased weights of thyroids (absolute and relative), liver (absolute and relative) and pituitary (relative), decreased serum T4 levels, and decreased thyroidal iodine uptake, and organification were observed in TU and TU+VA groups, as compared to the control group. Thyroidal iodine organification was also decreased in VA group. However, there were no differences in these parameters between TU and TU+VA groups. No treatment related effects were noted in serum T3 and TSH. No remarkable differences were observed in the histopathological findings of thyroids, liver, and pituitary between TU and TU+VA group, although diffuse follicular cell hyperplasia was induced in these groups. The results in the present study suggest that the enhancement effect of VA on thyroid proliferative lesions may not be attributable to the modification of thyroidal iodine uptake.

The extensive worldwide efforts of structural modification of natural pyrethrins for better performances have resulted in successful development of a wide variety of synthetic pyrethroids with tremendously high efficacy, knock-down activity or vapor action, and/or with acceptable environmental stability and safety. Currently these pyrethroids including their preferentially manufactured stereoisomers are widely used in agriculture, and for public health as well as household insect control. The detailed toxicology and metabolism studies intended to attain human risk assessment have revealed that with voltage-dependent sodium channel as target site pyrethroids induce pronounced repetitive activity characterized grossly by tremor, hypersensitivity, choleoathetosis, and salivation. In addition, so-called cyano-pyrethroids cause transient skin paresthesia in workers. With regard to tumorigenicity, mutagenicity, teratogenicity and developmental toxicity, no significant findings have been reported. Pyrethroids are eliminated from the animals quite rapidly and completely, undergoing oxidation and ester hydrolysis followed by various conjugations, with low tissue residues. Thus, overall, sound scientific bases exist for human risk assessment under the present usage conditions.

We examined whether the repeated oral administration with a high dose of l -3-(3,4-dihydroxyphenyl)-alanine ( l -DOPA) in 0.5% carboxymethyl cellulose increases serum luteinizing hormone (LH) levels in male rats. Serum LH levels were increased 4 h after a single administration of 1000 mg/kg l -DOPA to male rats, and returned to control levels within 8 h after administration. Four hours after a single administration, serum LH levels were significantly increased by l -DOPA at 1000 mg/kg, but not at 20, 100 or 200 mg/kg. Decreases in body weight and relative weight of the prostate were observed after 7 and 14 days of administration of 1000 mg/kg per day l -DOPA, but no changes were observed in weight of the testis, epididymis or seminal vesicle. The administration of l -DOPA at 500 or 1000 mg/kg per day for 7 or 14 days resulted in increased basal serum LH levels and decreased basal serum prolactin levels 24 h after the last administration. Serum testosterone levels tended to be higher in treated than in control rats. The levels of two metabolites of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in rats treated with 500 mg/kg per day tended to be slightly higher than those in control rats after 7 days of administration. Levels of DA, DOPAC and HVA were significantly increased after 7 and 14 days of administration of 1000 mg/kg per day and after 14 days of administration of 500 mg/kg per day. The level of norepinephrine, but not its metabolite 3-methoxy-4-hydroxyphenylglycol, was significantly increased after only 7 days of administration of 1000 mg/kg per day. No significant changes were observed in levels of 5-hydroxytryptamine or its metabolite 5-hydroxyindole-3-acetic acid with administration of 500 or 1000 mg/kg per day. These findings suggest that a prolonged treatment with a high dose of l -DOPA in male rats induces release of LH from the pituitary, resulting in sustained elevation of LH levels in peripheral circulation.

Studies were performed to examine the mechanism by which testicular Leydig cell tumors are induced in rats by administration of the antimicrobial agent oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid). In these studies, the effects of oxolinic acid on serum levels of luteinizing hormone (LH), testosterone, and prolactin and the binding of testosterone to prostatic androgen receptors were examined. In a long-term hormonal study, male Wistar rats were fed a diet containing oxolinic acid at 0, 100, 1000, or 3000 ppm for 104 weeks. A statistically significant increase in serum LH levels was observed at 1000 and 3000 ppm, but no dose of oxolinic acid had a significant effect on serum testosterone levels. Serum LH levels were no longer elevated above control levels within 2 weeks of cessation of the administration of oxolinic acid. Oxolinic acid was found to have no effect on the rate of clearance of exogenous LH from the circulation. Serum prolactin levels were decreased by the administration of oxolinic acid. The increase in serum LH induced by oxolinic acid was completely blocked by the intraperitoneal injection of the dopamine antagonist haloperidol (2 mg/kg). In addition, no significant affinity of oxolinic acid for androgen receptors was found in an in vitro study. These findings suggest that: (1) oxolinic acid induces Leydig cell tumors in rats by chronically stimulating the release of LH from the pituitary, (2) the mechanism of stimulating the release of LH involves facilitation of the dopaminergic systems in the hypothalamus-pituitary axis, and (3) oxolinic acid has no effect on androgen-mediated feedback inhibition.

The distribution of glutathione S-transferase (placental form; GST-P) and the c-myc gene product in rat hepatic preneoplastic lesions induced by diethylnitrosamine (DEN) and clofibrate was examined immunohistochemically. An in vivo medium-term assay system (4) was used to study the effects of chemical hepatocarcinogenesis. Rats initially received a single intraperitoneal dose (200 mg/kg) of DEN, and after week 2,1% clofibrate in diet for consecutive days. The rats were subjected to partial hepatectomy at week 3 and sacrificed at weeks 8, 20 and 30

Hyaline droplets in kidney tubules of rats with chronic progressive nephropathy were histopathologically compared with those found in 2, 2, 4-trimethylpentane (TMP) induced α2u-globulin nephropathy. Repeated oral administration of TMP at the dosage of 50mg/kg/day for four weeks caused development of hyaline droplets which were all immunohistochemically positive for α2u-globulin. In control rats (13 to 52 weeks-old), two types of hyaline droplets were observed: one small round and multiple type being deeply eosinophilic and refractile (hyaline droplet type); and the other being slightly eosinophilic, large, and generally single (eosinophilic body type). Both were positive for anti-α2u-globulin immunohistochemical staining. Fifty-two and 109 weeks-old male rats with chronic progressive nephropathy showed “hyaline droplet degeneration” of tubules with hyaline cast formation in the lumina. Immunohistochemical examination revealed such “hyaline droplet degeneration” to be positive for albumin, but negative for α2u-globulin. Under the electron microscope, round, amorphous, and moderately electron-dense phagolysosomes were observed in the tubules of “hyaline droplet degeneration” cases, clearly different from the polyangular and crystalline-like electron-dense phagolysosomes of normal α2u-globulin reabsorption droplets in young rats or TMP induced α2u-globulin accumulation droplets. The present results suggest that the etiology of chronic progressive nephropathy may be somewhat different from that of α2u-globulin induced nephropathy in terms of the causative protein.

To examine the mechanism and toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diethofencarb (isopropyl 3, 4-diethoxycarbanilate), male Sprague-Dawley rats were fed diethofencarb in diets at concentrations of 0, 5, 000 or 20, 000 ppm for 3 months. Examinations mainly for thyroid functions including thyroid uptake of &lt125&gtI, serum thyroid hormone and thyroid stimulating hormone (TSH) level, hepatic UDP-glucuronyltransferase (UDP-GT) activity and histopathological examination in thyroid were performed at week 13. Decreases of body weights and food consumptions were observed at and above 5, 000 ppm. Under these conditions, decrease of serum free T4 and increase of serum TSH level were observed only at 20, 000 ppm, concurrently with liver weight increase at and above 5, 000 ppm and increase of hepatic UDP-GT activity at 20, 000 ppm. However, no compound related effects were noted in thyroid weight, thyroid uptake of &lt125&gtI and gross or histopathological examination in thyroid. These results indicate that the administration of diethofencarb leads to an increase in UDP-GT activity and acceleration of thyroid hormone excretion from the liver. The acceleration causes a decrease in serum free T4 level, triggering the feedback mechanism of the pituitary gland, promotion of TSH release and consequently an increase in serum TSH level. Thus, the slightly higher incidence of thyroid follicular cell tumors observed in the chronic and oncogenicity study with non-genotoxic diethofencarb is considered to be caused by these weak pituitary-thyroid hormonal imbalances. The toxicological significance in humans is extremely low according to the well established facts that the chronic TSH stimulatin would not induce thyroid tumors in humans and humans may be less sensitive than rats in regard to the response to goitrogenic stimuli.

In recent years, mode of action (MOA) frameworks have been developed through the International Life Sciences Institute Risk Science Institute and the International Programme on Chemical Safety, including an evaluation of the human relevance of the animal MOA data. In the present paper, the MOA for rat liver tumors induced by Metofluthrin is first analyzed through this framework based on data from studies on Metofluthrin and information on related chemicals from the literature. The human relevance of the rat liver carcinogenic response is then discussed based upon the human relevance framework. Two-year treatment with high dose of Metofluthrin produced hepatocellular tumors in both sexes of the Wistar rats. Metofluthrin induced CYP2B (increased smooth endoplasmic reticulum), resulted in increased liver weights which were associated with centrilobular hepatocyte hypertrophy, and induction of increased hepatocellular DNA replications. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. Furthermore, CYP2B induction by Metofluthrin was shown to involve activation of the constitutive androstane receptor in rat hepatocytes. Based on the evidence, including a comparison with the results with another chemical, phenobarbital, acting by a similar MOA, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans.

Two-year treatment with high doses of Metofluthrin produced hepatocellular tumors in both sexes of Wistar rats. To understand the mode of action (MOA) by which the tumors are produced, a series of studies examined the effects of Metofluthrin on hepatic microsomal cytochrome P450 (CYP) content, hepatocellular proliferation, hepatic gap junctional intercellular communication (GJIC), oxidative stress and apoptosis was conducted after one or two weeks of treatment. The global gene expression profile indicated that most genes with upregulated expression with Metofluthrin were metabolic enzymes that were also upregulated with phenobarbital. Metofluthrin induced CYP2B and increased liver weights associated with centrilobular hepatocyte hypertrophy (increased smooth endoplasmic reticulum [SER]), and induction of increased hepatocellular DNA replication. CYP2B1 mRNA induction by Metofluthrin was not observed in CAR knockdown rat hepatocytes using the RNA interference technique, demonstrating that Metofluthrin induces CYP2B1 through CAR activation. Metofluthrin also suppressed hepatic GJIC and induced oxidative stress and increased antioxidant enzymes, but showed no alteration in apoptosis. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. All of these effects were reversible upon cessation of treatment. Metofluthrin did not cause cytotoxicity or peroxisome proliferation. Thus, it is highly likely that the MOA for Metofluthrin-induced liver tumors in rats is through CYP induction and increased hepatocyte proliferation, similar to that seen for phenobarbital. Based on analysis with the International Life Sciences Institute/Risk Science Institute MOA framework, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans, at least at expected levels of exposure.

In the present study, susceptibility of CB6F1 mice carrying the human prototype c-Ha- ras gene (rasH2 mice) and p53 gene knockout mice ( p53 (+/−) mice) to urethane-induced lung carcinogenesis was compared under the same experimental conditions. Both strains were administered 500 ppm urethane in their drinking water for 3 weeks. At week 26, lung adenocarcinomas and adenomas were observed in 53% and 100% of rasH2 mice, respectively, and lung adenomas were observed in 67% of rasH2 littermate (non-Tg) mice. However, lung tumors were not observed in either p53 (+/−) or p53 (+/+) mice. Peliosis hepatis and hepatic hemangiomas were observed in 27% and 67% of p53 (+/−) mice, but only in 6.7% and 6.7% of the rasH2 animals, respectively. Under the same experimental conditions, BALB/c mice, the strain of origin of the rasH2 mice, developed lung adenomas at an incidence of 93%, whereas none of the C57BL/6 original strain for p53 (+/−) mice developed lung tumors. Peliosis hepatis was observed in 40% of the C57BL/6 mice, but not in BALB/c mice; hepatic and splenic hemangiomas were not observed in these animals. These results indicate that organ susceptibility of rasH2 and p53 (+/−) mice is inherited from their strains of origin, the rasH2 and BALB/c lines being much more sensitive to the induction of pulmonary carcinogenesis.

As part of an international validation project to establish a test protocol for the 'Enhanced OECD Test Guideline no. 407', a 28-day repeated dose study of flutamide was performed (1) to examine which of the current and/or additional parameters can detect endocrine effects of test chemicals most reliably and sensitively, (2) to investigate whether it is actually feasible to routinely include all additional parameters into the testing routine, and (3) to assess intra-laboratory variability by performing two identical studies (experiments A and B) in parallel using groups of five animals each per dose and sex. Groups of five male and five female CD(SD)IGS rats were treated by oral gavage with 0, 1, 10 and 100 mg flutamide/kg body weight for at least 28 days. The dose level considered to be around the MTD (100 mg/kg) exerted the expected antiandrogenic effects on androgen related tissues: significant decrease of the weights of androgen dependent organs and the sperm count and increase in histopathological lesions. At the middle dose (10 mg/kg), significant decrease of prostate weight (ventral and dorso-lateral parts combined) was observed and it was suggested that weight measurement of androgen dependent organs provides the most reliable and sensitive endpoint with this protocol. As for the feasibility, because of many items in this protocol, selection should be based on the sensitivity. From our data, addition of weight measurement of androgen dependent organs to the items of the existing OECD 407 guideline might allow accurate screening for endocrine disruptors. At the dose level considered to be around the MTD, the findings achieving statistical significance in one experiment with five animals/dose/sex could be reproduced in the second experiment, and evaluation with the small groups was consistent with findings using the combined groups of 10 animals/dose/sex. The results demonstrate that the protocol can reliably detect antiandrogenic effects of flutamide.

Pregnant rats were administered flutamide (0 and 10 mg/kg, p.o.) from gestation Day 14 to post-parturition Day 3 and effects on responsiveness to androgens (testosterone propionate, TP; dihydrotestosterone, DHT) in male offspring were examined with a Hershberger assay. Male pups of each group were assigned to 6 subgroups as follows: Group 1, castration and euthanized at postnatal Day 46 (PND 46); Group 2, castration + vehicle; Group 3, castration + TP; Group 4, castration + DHT; Group 5, vehicle; Group 6, DHT. After castrations were conducted at PND 36, animals were treated with TP (2 mg/kg in corn oil, s.c.) or DHT (1.25 mg/kg in corn oil, s.c.) once a day for 10 days, beginning at PND 46. At PND 56, the following organs/tissues were removed and weighed: ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani muscle plus bulbocavernosus muscle, Cowper's gland, and glands penis. Analysis of serum testosterone, LH and FSH in Groups 2, 3, 4, 5 and 6, and RT-PCR using prostate tissue from Groups 2, 3 and 4 were carried out. Perinatal exposure to flutamide caused decreased weights of androgen-dependent organs. Responses to androgens were recognized in organs of all castrated groups, with increased organ weights, especially in animals administered TP where values were essentially equal to or greater than those of intact animals in both the control and the 10 mg/kg group. On the other hand, the degree of weight increase of the ventral prostate and seminal vesicles with TP or DHT treatment in castrated animals was smaller in the flutamide administration group than in the controls. In hormone assays, castrated + vehicle animals showed higher serum LH than the other groups. Serum FSH was high in the castrated groups (Group2>Group 4>Group3), while in the noncastrated group a constant level was noted, with or without flutamide. No effect of flutamide administration was observed regarding sex hormone. RT-PCR using ventral prostate tissue revealed no significant differences in expression of AR, C3, VEGF, TGF-beta1, beta2, KGF and CK8 mRNA after androgen treatment between the control and flutamide treatment groups. C3 mRNA was increased in androgen-treated animals, whereas AR, TGF-beta and KGF mRNAs were decreased. Perinatal exposure to anti-androgen causes irreversible abnormalities in male pups. Concerning the responsiveness to TP and DHT, the degrees of weight changes in ventral prostate and seminal vesicles in castrated animals were decreased. However, the other organ weights, the sex hormone levels and androgen-reactive gene expression in the ventral prostate were not influenced by perinatal flutamide treatment in the present study.

Synthetic pyrethroids are among the most common insecticides and pesticides currently in use worldwide. Recently, d -phenothrin, a synthetic pyrethroid, is suspected to have endocrine activities through the estrogen and androgen receptors. However, no study has been conducted to evaluate its potential for hormonal activity using an in vivo test specifically focused on estrogenic and androgenic activities. In this study, we evaluated the interaction of d -phenothrin (0, 100, 300 or 1000 mg/kg per day, p.o.) with estrogen- or androgen-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the uterotrophic and Hershberger assays have not yet been fully developed, both are widely used and are being considered by the OECD as short-term screening assays for hormonal activity. The highest dose level tested for d -phenothrin was a limit dose (1000 mg/kg per day) designated in the current draft protocol by the OECD, and in fact there was no excessive systemic toxicity in both assays; slightly increased liver weight but no change of serum androgen levels in accessing anti-androgenicity. Potential estrogenic effect of d -phenothrin was evaluated by means of 3-day uterotrophic assay using immature Crj:CD(SD)IGS rats (20 days of age). No increase in uterine weight (wet or blotted) was observed following oral exposure to d -phenothrin. Reference control ethynyl estradiol (0.001 mg/kg per day) showed a significant effect in this assay protocol. A 10-day Hershberger assay using castrated peripubertal male rats measures the androgenic or anti-androgenic effects of the test chemicals on several accessory glands/tissues (the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscles, glans penis and Cowper's glands). d -Phenothrin was administered by oral gavage for 10 days to castrated male Crj:CD(SD)IGS rats (7 weeks of age, rats were castrated at 6 weeks of age) with or without co-administration of 0.2 mg/kg per day testosterone propionate (subcutaneous injection on the dorsal surface). Reference controls of methyltestosterone and p , p ′-DDE (100 mg/kg per day) provided significant effects in this assay protocol, whereas d -phenothrin did not show any androgenic or anti-androgenic effects. It is concluded that, based on the results of these two reliable in vivo assays, d -phenothrin exhibits no potential to cause adverse estrogenic or (anti-)androgenic effects even at dose of 1000 mg/kg per day, the limit dose designated in the current draft protocol by the OECD.

Synthetic pyrethroids are among the most common pesticides and insecticides currently in use worldwide. Recently, chemicals classified as synthetic pyrethroids are suspected as being endocrine disrupting chemicals. However, no study has been conducted to assess their potential hormonal activities using in vivo test specifically focused on endocrine disruption. In the present study, we evaluated the interaction of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) with androgen receptor (AR)- and estrogen receptor (ER)-mediated mechanisms using in vivo short-term assays. While internationally standardized protocols for the Hershberger and uterotrophic assays have not yet been fully developed, both are widely used and are being considered by OECD as short-term screening assays for hormonal activity. A 5-day Hershberger assay using castrated male rats measures agonistic and androgenic ability of the test chemicals to AR of several accessory glands/tissues (the ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands, and levator ani plus bulbocavernosus muscles). Esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (25, 50, or 75 mg/kg/day) was administered by oral gavage for 5 days to castrated male Crj:CD(SD)IGS rats (1 week after the castration, 11 weeks of age) with or without coadministration of 0.25 mg/kg/day testosterone propionate (subcutaneous injection on the dorsal surface). The highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. None of esfenvalerate, fenvalerate, and permethrin showed any androgenic or antiandrogenic effects. Reference control of p,p'-DDE and methyltestosterone (100 mg/kg/day) provided significant effects in this assay protocol. Potential effects of these pyrethroids mediated through the ER were evaluated by means of 3-day uterotrophic assay using ovariectomized Crj:CD(SD)IGS rats (2 weeks after the ovariectomy, 8 weeks of age). No increase in weight of uterus (wet or blotted) was observed following oral exposure to esfenvalerate (5, 10, or 20 mg/kg/day), fenvalerate (20, 40, or 80 mg/kg/day), or permethrin (37.5, 75, or 150 mg/kg/day), respectively. Again, the highest dose levels tested for each chemical were considered the maximum level that could be used without causing excessive systemic toxicity. Reference controls consisting of ethynyl estradiol (0.03 mg/kg/day) and methoxychlor (125 mg/kg/day) both showed a significant effect in this assay protocol. It is concluded that, based on the results of these two reliable in vivo assays, none of esfenvalerate, fenvalerate, or permethrin exhibit any potential to cause adverse (anti-) androgenic or estrogenic effects at dose levels below that of those causing excessive systemic toxicity.

To obtain information on the effects of nongenotoxic carcinogens at low doses for human cancer risk assessment, the carcinogenic potential of the organochlorine insecticide, 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), in the liver was assessed in F344 rats. In experiment 1, 240 male animals, 21 days old, were administered 0, 0.5, 1.0, 2.0, 5.0, 20, 100 and 500 ppm DDT in the diet for 16 weeks. Experiment 2 was conducted to elucidate the carcinogenic potential of DDT at lower levels using 180 rats given doses of 0, 0.005, 0.01, 0.1, 0.2 and 0.5 ppm. The livers of all animals were immunohistochemically examined for expression of glutathione S-transferase placental form (GST-P), putative preneoplastic lesions. Quantitative values for GST-P-positive foci in the liver were increased dose-dependently in rats given 20 ppm DDT and above with statistical significance as compared with the concurrent control value. In contrast, doses of 0.005 and 0.01 ppm were associated with a tendency for decrease below the control value, although not significantly. Western blotting analysis show that cytochrome P-450 3A2 (CYP3A2) protein expression tended to decrease at 0.005 and 0.01 ppm, a good correlation being observed with the change in the number of GST-P-positive foci. These findings suggest that a DDT hepatocarcinogenicity may show nonlinear response, that is, hormetic response at low doses. Furthermore, since CYP3A2 protein expression appears to be important for the effects of phenobarbital and the α-isomer of benzene hexachloride, mRNAs for IL-1 receptor type 1 (IL-1R1) and TNF-α receptor type 1 (TNFR1) whose ligands have roles not only in downregulating CYP3A2 expression but also in inducing antiproliferative effect or apoptosis in hepatocyte were examined. Increase was observed at low doses of DDT. Oxidative stress in liver DNA, assessed in terms of 8-hydroxydeoxyguanosine as a marker, was also decreased. These findings suggest that the possible hormetic effect that was observed in our detailed low-dose study of DDT carcinogenesis, although not statistically significant, may be linked to levels of oxidative stress and proinflammatory cytokines. © 2002 Wiley-Liss, Inc.

The rodent Hershberger assay has been used predominantly by the pharmaceutical industry to evaluate androgenic and antiandrogenic chemicals for potential therapeutic use. However, this assay has not yet been formally validated and standardized for use in toxicology testing. There are many variations in the protocol used for this assay. The weight of androgen-dependent tissues is a definitive endpoint in the Hershberger assay. To find out the reliable assay protocol with feasibility, although many possible factors may affect assay reliability, the present study consist of a series of three separate experiments focused on method of dissection and weighing of accessory sex glands (ASGs: ventral and dorso-lateral prostate, seminal vesicles together with coagulating glands, and Cowper's glands), animal age and number of doses. Furthermore, male pubertal assay, an alternative to the Hershberger assay, was also examined its reliability. Experiment 1 explored whether reliably accurate ASG weights can be obtained after formalin fixation. The ASGs were collected from castrated male rats (11 weeks of age) treated daily with corn oil, or testosterone propionate (TP, 0.25 mg/kg/day, s.c.) and p,p′-DDE (0 or 100 mg/kg/day, p.o.) for 5 days. One day after the final treatment, the ASGs were removed carefully and weighed separately, and then fixed overnight in a 10% neutral-buffered formalin and weighed again. After that, the tissues were dried overnight in an oven and weighed again. A high correlation between fresh and fixed tissue weights, and a high correlation between fixed and dried tissue weights were noted. The changes in the tissue weight due to fixation were marginal and were proportional to the fresh weights of the individual tissue. Standard deviation of the fixed tissue weight in each group and the magnitude of responses to TP or p,p′-DDE in fixed tissues were equivalent to those in fresh or dried tissues. These findings indicate that formalin fixation does not interfere with interpretation of assay results, and this procedure was used in the subsequent experiments. Experiments 2 and 3 explored whether animal age or treatment duration altered assay sensitivity. In Experiment 2, antiandrogenic effect of p,p′-DDE (100 mg/kg/day) was detected after 5-and 10-day treatment irrespective of animal age (7 vs 11 weeks). In Experiment 3, antiandrogenic effects of flutamide (1 and 10 mg/kg/day) and p,p′-DDE (10 and 100 mg/kg/day) were compared between two different protocols, the 10-day assay using peripubertal rats and the 5-day assay using young mature rats. Results demonstrated that both protocols could significantly detect antiandrogenic effects of flutamide and p,p′-DDE. These findings demonstrate that (1) dissection and weighing of ASGs after formalin fixation is reliable in the Hershberger assay, (2) when this procedure is used, the 5-day Hershberger assay using young mature rats, expected to be more feasible assay than the 10-day assay using peripubertal rats, is also reliable as well as the 10-day assay using peripubertal rats. Furthermore, we confirmed that male pubertal assay with use of dissection and weighing of fixed tissues also reliable.

A 5-day Hershberger assay utilizing mature male rats and a pubertal male assay were evaluated for the ability to detect antiandrogenic compounds such as flutamide, an androgen receptor antagonist. Six days after the operation, implantation with two silicon capsules containing testosterone (T) (30 mg/capsule) in castrated rats provided the ventral prostate and seminal vesicle weights as well as serum T and luteinizing hormone (LH) levels equivalent to those of the controls (non-castrated, non-implanted rats). Castrated rats implanted with two T-capsules (6 rats/dose) were treated by gavage for 5 days with vehicle (0.5% carboxymethylcellulose) or flutamide (0.15, 0.6, 2.5, or 10 mg/kg/day). Flutamide produced significant decreases in weights of the seminal vesicles and the levator ani plus bulbocavernosus muscles (> or =0.6 mg/kg/day) and ventral prostate (> or =2.5 mg/kg/day), and an increase in serum LH levels (> or =2.5 mg/kg/day), but no changes in serum T levels. When age-matched intact male rats were treated with 10-mg/kg/day flutamide, a significant increase in serum T levels was observed concomitant with a tendency of increased LH. The organ weights were also decreased; however, the changes were less than those in the castrated, T-implanted rats. Immature intact male rats (10 rats/dose) were treated for 20 days with flutamide (0, 0.15, 0.6, 2.5, or 10 mg/kg/day). Flutamide produced significant decreases in weights of the seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles at 2.5 and 10 mg/kg/day. Serum LH levels, but not T levels, were increased at 10 mg/kg/day. Statistical significance of some of these changes was not observed in the 6 animals/dose examined. Our findings support that the Hershberger assay, in the current conditions, is the most sensitive among the assays examined and a useful short-term screening method for the detection of antiandrogenic compounds.

To examine whether acute dystonia is induced by neuroleptic treatment, common marmosets were treated with haloperidol orally twice a week over 25 weeks until dystonic behavior was elicited. Movement disorders such as acute dystonia were observed 6 weeks after the initial treatment, and had appeared in all treated animals by 25 weeks. Once these movement disorders were induced, they consistently reappeared after further treatment with haloperidol, and once haloperidol dosing was discontinued, the episodes vanished. Then, various neuroleptic drugs (bromperidol, chlorpromazine, risperidone thioridazine, sulpiride, tiapride, and clozapine) or a nonneuroleptic drug (diazepam) were administered orally instead of haloperidol in the above animals. All the neuroleptic drugs except for clozapine elicited similar abnormal behavior, while diazepam failed to induce any dystonia. An anticholinergic drug, trihexyphenidyl, which is known to reduce acute dystonia in patients, was also given orally to the above haloperidol-sensitized animals, followed by further treatment with haloperidol 30 min later. This clearly suppressed the induction of dystonia by haloperidol. The similarity between these findings for haloperidol-pretreated common marmosets and clinical findings suggests that the present model is useful for predicting the potential of antipsychotics to induce acute dystonia in humans.

Oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid), an antimicrobial agent, raises the serum levels of luteinizing hormone (LH) and increases the incidence of testicular Leydig cell tumors in male rats. In the present study the mechanism by which serum LH levels are raised in male rats receiving oxolinic acid was investigated. Aged Wistar rats were fed a diet containing oxolinic acid at 0 or 3000 ppm for more than 4 weeks. There was no effect of oxolinic acid on either the maximal levels of serum LH after castration nor on the serum levels of LH stimulated with 1 μg/rat of luteinizing hormone-releasing hormone (LHRH). The concentrations of testosterone in serum and testis were not changed by the treatment of oxolinic acid. In the in vitro organ culture, the testes of rats receiving oxolinic acid released testosterone in the same manner as the controls, in the presence or the absence of human chorionic gonadotropin (100 mIU/ml). The oxolinic acid-stimulated serum LH was not increased further by the daily administration of L-dopa (500 mg/kg/day, po, 7 days) and was blocked by the injection of a dopamine antagonist, haloperidol (2 mg/kg, ip). In a microdialysis study, oxolinic acid increased the extracellular concentration of dopamine in the preoptic area of hypothalamus. These findings suggest that a high dietary level of oxolinic acid elevates LH release from the anterior pituitary with an increase in LHRH, in part, by the excitatory input of a dopaminergic system in the preoptic area of rat hypothalamus.

A chronic feeding study was conducted to determine the carcinogenic potential of oxolinic acid, an antimicrobial agent, in rats and mice. Oxolinic acid was administered in the diet to Wistar rats (0, 30, 100, 300 or 1000 ppm; 50 rats/dose/sex) for 104 wk and to ICR mice (0, 50, 150 or 500 ppm; 50 mice/dose/sex) for 78 wk. Clinical signs, body weight, food consumption, autopsy findings and histopathological data were noted. Mortality was unaffected by oxolinic acid administration in neither species. In rats, body weight gain was suppressed in both sexes at 1000 ppm. Histopathological examinations conducted after autopsy at 104 wk revealed a slight increase in benign Leydig cell tumours of the testis at 1000 ppm. which did not appear until late in the lifetime of rats. No other treatment-related neoplastic lesions were observed in rats. Non-neoplastic lesions in males at 1000 ppm included Leydig cell hyperplasia and tubular atrophy of the testes. In mice, decreased body weight gain was observed in both sexes at 500 ppm, but no non-neoplastic or neoplastic lesions attributable to the treatment with oxolinic acid occurred in either sex. In conclusion, oxolinic acid induced benign Leydig cell tumours of the testis in rats at the highest dose level tested (1000 ppm). The no-effect level for tumour induction was confirmed to be 300 ppm (10.9 mg/kg/day) in rats. None was induced in mice.

To investigate the mechanism and toxicological significance of testicular interstitial cell tumors (ICT) observed in a long-term rat study with procymidone, N-(3, 5-dichlorophenyl)-1, 2-dimethylcyclopropane-1, 2-dicarboximide, male Sprague-Dawley rats were fed procymidone in diets for up to 6 months with a positive control group receiving a single subcutaneous injection of cadmium chloride. Examinations mainly for gonadal functions such as serum testosterone and luteinizing hormone (LH), reproductive organ weight and histopathology presented evidence of the indirect involvement of gonadotropins in the production of ICT in rats. A significant increase in both serum testosterone and LH was observed in the early stage at high dietary concentrations of procymidone without any lesion in gonadal systems in histopathology, whereas administration of cadmium chloride produced the expected substantial increase in serum LH and a concomitant decrease in serum testosterone with a marked damaging effect on gonadal systems. Increases in serum testosterone and LH levels in animals receiving procymidone were reversible. The no-effect level for procymidone on serum testosterone and LH was 300 ppm over six months of treatment. The possible mechanism of ICT production in rats by non-genotoxic procymidone, structurally similar to flutamide, a synthetic non-steroidal antiandrogen, is likely to be derived from its induction of a hypergonadotropism due to the competitive binding to the androgen receptor, preventing the normal effect of testosterone to control the circulating level of LH.

Wistar rats and ddY mice were fed diet containing 10 to 3000 ppm technical fenvalerate for the major portion of their life span. Microgranulomatous changes were observed in lymph nodes, liver and spleen of both rats and mice as well as in adrenals of rats. The no-effect level for microgranulomatous changes was 30 ppm for mice and 150 ppm for rats. Transmission electron microscopy of phagocytes in the granulomatous foci revealed needle- or rod-shaped crystalline bodies within the cytoplasm. To examine reversibility of the microgranulomatous changes, ddY mice were fed the diet containing technical fenvalerate at the levels of 1000 and 3000 ppm for 6 weeks, followed by rearing on the control diet for up to 12 months. The size and number of the microgranulomatous changes were reduced with time. The microgranulomatous changes were typical of foreign body granulomas; they did not have the appearance of granulomas formed in response to immunologic stimulus.

Male mice of the ddY strain were fed a diet containing the [2S, alpha S]-, [2S, alpha RS]-, [2R, alpha S]-, and [2R, alpha R]-isomers of fenvalerate. Microgranulomatous changes were observed only in mice treated with the [2R, alpha S]-isomer at 125 and 1000 ppm for 1, 2, or 3 months. In contrast, the changes did not occur in mice treated with the [2R, alpha R]-isomer under the same conditions. Feeding of the [2S, alpha S]- and [2S, alpha RS]-isomers for 1 year did not cause the microgranulomatous changes at 500 or 1000 ppm. To clarify the causative agent of granuloma formation, cholesterol ester of 2-(4-chlorophenyl)isovaleric acid (CPIA), a lipophilic conjugate from the [2R, alpha S]-isomer of fenvalerate, was injected iv into ddY mice. Microgranulomatous changes were observed in the liver of mice treated with the [2R]-, [2S]-, or [2RS]-CPIA-cholesterol ester 1 week after a single treatment of 1, 10, or 100 mg/kg, as well as in liver of mice treated with a single dose of 10 or 30 mg/kg of the [2R]-CPIA-cholesterol ester and kept up to 26 weeks afterward. Histochemistry and microscopic autoradiography of the liver of mice demonstrated the presence of tritium derived from 3H-labeled[2R]-2-(4-chlorophenyl)isovalerate and cholesterol. Histochemistry also was positive for cholesterol ester in livers of mice treated with the [2R, alpha S]-isomer of fenvalerate. These results lend support for the hypothesis that CPIA-cholesterol ester is the causative agent of microgranulomatous changes induced by fenvalerate.

1. The substrate specificity of microsomal carboxyesterase(s) responsible for the formation of cholesteryl [2R]-2-(4-chlorophenyl) isovalerate from fenvalerate was investigated by incubating mouse kidney microsomes with 14C-cholesterol and the following substrates: fenvalerate isomers, fenvalerate analogues, other pyrethroids, methoprene and cycloprate analogues. Among the four isomers of fenvalerate, only the [2R, alpha S]-isomer yielded a cholesterol ester, being identical with the result obtained in the in vivo study. Some fenvalerate analogues produced cholesterol ester conjugates, but no other pyrethroids nor methoprene produced such conjugates. Some cycloprate analogues gave the corresponding cholesterol ester, the yields of which were dependent on their carbon-chain lengths. 2. Cholesterol ester formation in vitro from these fenvalerate analogues was well correlated with granuloma formation observed when the analogues were given to mice at 3000 ppm for a month. 3. Steroids other than cholesterol were also investigated as acceptors of the acid moiety of the [2R, alpha S]-isomer by incubating solubilized carboxyesterase(s) with the [2R, alpha S]-isomer in the presence of egg lecithin and several steroids. Dehydroisoandrosterone and pregnenolone were found to give the corresponding ester conjugates.

雄ラットに四塩化炭素 (CCl4), 4, 4′-ジアミノジフェニルメタン (DDM), 低タンパク高脂肪 (LPHF) を12週間にわたって与えると, それぞれ肝に小葉中心性脂肪変性繊維化・肝硬変, 胆管増生・細胞滲潤, 実質細胞のび漫性脂肪変性を主特徴とする肝障害の発生がみられ, また血液, 肝の各生化学的パラメーターに変化を生ずる. これに対応するP-450, アニリン水酸化酵素, アミノピリン N-脱メチル酵素, p-フェニルアセテート水解酵素, グルタチオン S-アルキルトランスフェラーゼ活性の低下は対照に比較してDDM

14C-フェニル標識フェニトロチオンを0.5mg/kg/dayの割合で7日間, 日本ザーネン種の山羊に経口投与し, その代謝をしらべた. 最終投与1日後, 肝, 胃, 腸では0.812～1.164ppmの14Cが見いだされたが, 血液, 脳, 肺, 卵巣などその他の臓器の14Cは0.001～0.031ppmにとどまり, また, いずれの臓器および組織からもフェニトロチオンおよびフェニトロオキソンは検出されず, 胃および腸より微量のアミノフェニトロチオンが検出されるにすぎなかった. 14Cは体内より急速に消失し, 最終投与1週間後には全14Cの94% (尿50%, 糞44%) が, また, 残存14Cも時間の経過とともに漸次体内より排泄された. ミルク中には最高値0.011ppm (投与量の0.1%以下) の14Cが見いだされた. 尿, 糞およびミルク中の主要代謝産物はアミノフェニトロチオン, N-スルフォアミノフェニトロチオンおよびN-アセチルアミノフェニトロオキソンのO-脱メチル体であった. フェニトロチオンおよびフェニトロオキソンは見いだされなかった.反芻動物においては, フェニトロチオンはまず, ニトロ基の還元をすみやかに受け, その後アミノ基の抱合, P=SのP=Oへの酸化, O-脱メチル化などを経て, 分解すると推定された.

While the optical isomers of EPN were equally toxic to mice, (+)-EPN was 2.9 fold and 4.0 fold more toxic to houseflies and rice stem borer larvae, respectively, than the (-)-isomer. In addition, (-)-EPN produced paralysis of the legs in hens about 10 to 14 days after dosing, whereas (+)-EPN caused no paralytic effects. Thus, (+)-EPN appears to be a more appropriate insecticide than the racemic compound, since it combines high toxicity to insects and no delayed neruotoxicity in hens.