Analysis of classical life-time rodent bioassays conducted over the past 20 years under conditions of Good Laboratory Practice Regulations has led to an improved understanding of possible factors involved in oncogenesis in humans. Improvements in study design coupled with more detailed pathological examinations have led to new insights whereby neoplastic processes can be more clearly identified and understood with confidence in animal models. Improvements in the ability to identify potential risks through continued animal testing combined with incorporation of advances in genetics and molecular biology in elucidating mechanistic factors will greatly facilitate future research efforts to identify causative agents of human cancer. A great deal of attention will have to be paid to the concept of threshold doses and exposure levels that may be required to achieve preneoplastic conditions or the induction of carcinogenic processes. Future concerns of hazards, risk assessment and the evaluation of carcinogenic potential will involve an amalgamation and understanding of radiological and biological events, especially those of carcinogenic microorganisms, the effects of food and air borne carcinogens, effects of pollution, exposure to xenobiotics and imbalances and disruption of normal biologic functioning of endogenous physiologically active substances, to achieve a better understanding and ultimate prevention of human cancer.
We investigated the accumulation of CPT-11 and its metabolite(SN-38)in various organs and toxicities on multiple injections of CPT-11 under clinical regiments in SD rats. CPT-11(16.7 mg/kg equivalent to 100 mg/m2)was administered intravenously by a single injection, or by multiple injections in 1 course(once a week for three consecutive weeks)or 3 courses(1 course repeated 3 times at intervals of 2 weeks). There was no tendency for CPT-11 and SN-38 to accumulate in any organs regardless of the number of injections. Treatment-related changes were not observed in the general condition, body weight, hematology, biochemistry, and organ weights. Histopathological changes induced by CPT-11 were not persistent and the rats made a rapid recovery after the administrations. From these results, it is suggested that there is no toxicity caused by accumulation of CPT-11 and its active metabolite, SN-38, in organs under clinical regiments in rats.
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.
We previously reported that body weight on day 14 after birth in male offspring of rats given alkaline ionized water(AKW)was significantly heavier than that in offspring of rats given tap water(TPW), but no significant difference was noted in milk yield and in suckled milk volume between the two groups. Additionally, the offspring in the AKW group and TPW group were given AKW and TPW, respectively, at weaning, and unexpectedly, the necrotic foci in the cardiac muscle were observed at the 15-week-old age in the AKW group, but not in the TPW group. The present study was designed to clarify the factors which are involved in that unusual increase of body weight and occurrence of cardiac necrosis. Eight dams in each group were given AKW or TPW(control)from day 0 of gestation to day 14 of lactation. The milk samples were collected on day 14 of lactation and analyzed for concentrations of calcium(Ca), sodium(Na), potassium(K), magnesium(Mg)and chloride(Cl). The AKW and TPW were also analyzed. Ca, Na and K levels in milk were significantly higher in the AKW group compared to the TPW group. No significant difference was noted in the Mg and Cl levels between the two groups. These data suggested that the Ca cation of AKW enriched the Ca concentration of the milk and accelerated the postnatal growth of the off-spring of rats given AKW.
There is a possibility that serious liver dysfunction rarely observed in diabetic patients given troglitazone is attributable to idiosyncratic abnormalities in liver drug-metabolism. In addition, the results of blood biochemical examinations in serious cases of liver dysfunction showed a tendency for a high level of total bilirubin(T-Bil)over a long period compared with other indicators of liver dysfunction. Thus, we focused on genetic variation of UDP-glucuronosyltransferases(UGTs)that are involved in the conjugation of troglitazone and bilirubin. In this study, Gunn rats, which are hereditarily deficient in the UGT1 family of UGT isozymes, and Wistar rats, the parent strain of Gunn rats, were treated with troglitazone for 3 months at dose levels of 0, 100 or 400 mg/kg to investigate two possibilities:first, whether the genetic deficiency in UGT1s induces an alteration of the metabolic profile of troglitazone followed by liver dysfunction, and second, wheter the dosing of troglitazone to Gunn rats which sho hyperbilirubinemia result in liver dysfunction. As a result, the metabolic profile of troglitazone in Gunn rats was much the same as that of Wistar rats, suggesting that genetic deficiencies in UGT1s did not influence the metabolic profile of troglitazone. Moreover, no elevation of blood biochemical parameters, such as aspartate aminotransferase(AST)and alanine aminotransferase(ALT), or histopathological liver injuries, such as hepatocellular degeneration and necrosis, were observed in either strain of rats, and hyperbilirubinemia in Gunn rats was not aggravated by the dosing of troglitazone. These results strongly suggest that troglitazone was not metabolized by UGT1s but by other UGT isozyme(s)in rats, and that glucuronidation of troglitazone did not compete with glucuronidation of bilirubin in vivo. Thus, it is suggested that high levels of total bilirubin in patients with liver dysfunction induced by troglitazone are attributable to hypofunction due to hepatocellular injury, not to metabolic competition of bilirubin with troglitazone. Moreover, it is also suggested that the deficiency in the UGT1 family of UGT isozymes itself may not be the cause of liver dysfunction associated with troglitazone treatment.
UDP-glucuronosyltransferases(UGTs)involved in troglitazone glucuronidation in rats and humans have been characterized to support the previous toxicity study on troglitazone in Gunn rats and to examine whether the UGT polymorphism or inhibition of bilirubin metabolism is related to the clinically reported rare cases of liver failure. The experiments using Gunn rats revealed that UGT1 enzymes are not involved in troglitazone glucuronidation and that the responsible enzyme in rats was suggested to be UGT2B2, an androsterone UGT, by inhibition sudies. In humans, contribution of UGT1A1 was estimated to be about 30% of the total troglitazone glucuronidation by UGTs, using human liver microsomes and recombinant UGTs. Other UGT1 and UGT2 enzymes seem to be responsible for the rest of the troglitazone glucuronidation in humans. The multiplicity of UGTs involved in troglitazone glucuronidation in humans may allow even patients lacking bilirubin UGT(UGT1A1)activity to produce troglitazone glucuronide. These observations suggest that the polymorphism of UGT is not the reason behind the liver failure induced by the troglitazone treatment, and troglitazone does not inhibit bilirubin glucuronidation in clinical treatment. In addition, the increased bilirubin level in the blood of patients who have troglitazone-induced liver failure is a consequence of liver injury and not due to inhibition of bilirubin glucuronidation by troglitazone.
A comparison among rat sperm motility test methods including percent of motile sperm(% Motile), scoring method(Scoring), Ishii's method, Progressive Motility Test(PMT)and Sperm Quality Analyzer(SQA【○!R】), was conducted using data gathered from eleven laboratories. As a unified study design, mature male rats were orally treated daily for approximately 1 week with α-chlorohydrin(ACH), which is known to affect the sperm motility at the epididymis, at dose levels of 2.5, 5 and 10 mg/kg, and then subjected to more than two test methods for sperm motility in each laboratory. Scoring(4 or 5 grades), Ishii's method, PMT and SQA【○!R】 showed high sensitivity for the detection of the effects of ACH, which were not considered to be inferior to a computer-assisted sperm analyzer(CASA). Longer incubation time before testing was considered to contribute to detecting the effects of ACH. In particular, we realized that Scoring was a favorable method even if the demerit of poor objectivity was allowed for. Percent Motile showed lower sensitivity than other test methods. The differences in sensitivity between % Motile and other methods were considered to be based on whether the defects of progressive motion could be detected. Although % Motile cannot clearly judge whether immotile sperm are dead or alive, the value is a great help for the interpretation of the result from other methods. Based on the characters for detectability, objectivity and efficiency, the most suitable method of sperm motility should be selected according to the purpose of the toxicity study.