Screening a novel class of anticoagulants for multi-organ toxicity using Fischer 344 rats: involvement of clinical and anatomic pathologists in the early drug development process

BACKGROUND: Veterinary clinical and anatomic pathologists play a critical role in assessing the safety of new molecules. The process for evaluation of candidate molecules in drug discovery may vary markedly, depending on the unique characteristics of the compound class. OBJECTIVES: The goal of this report is to describe the evaluation process for assessing the potential toxicity of 2 anticoagulant compounds that were representative of molecules tested in early screening studies in Fisher rats, and to use these studies as an example of the strategic approach used by veterinary pathologists in pharmaceutical safety assessment. METHODS: Groups of 3 rats were given vehicle alone or one of several doses of compound A or B by oral gavage daily for 4 consecutive days. Survival; clinical signs; body and organ weight measurements; hematologic, coagulation, and clinical biochemical testing; and gross and histologic findings at necropsy were assessed. Transmission electron microscopy was used to characterize unique findings in the liver of rats treated with compound B. RESULTS: Both compounds caused dose-dependent prolongation of the prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin clotting time (TCT). Hepatobiliary and intestinal toxicity were identified by alterations in serum chemistry data, and by histopathologic findings. Electron microscopy and tissue inorganic phosphorus analysis revealed phospholipidosis in rats treated with compound B. CONCLUSIONS: Pharmacologically mediated or "on target" effects for these molecules were characterized by dose-progressive prolongation of the PT, APTT, and TCT. Nonpharmacologically mediated or "off-target" toxicity consisted of hepatoxicity and enterotoxicity. These liabilities required that scientists alter the original molecular scaffold to reach the desired therapeutic target and minimize toxicity.     

A risk assessment of atrazine use in California: human health and ecological aspects

A risk assessment of the triazine herbicide atrazine has been conducted by first analyzing the toxicity database and subsequently estimating exposure. Margins of safety (MOS) were then calculated. Toxicity was assessed in animal studies and exposure was estimated from occupational and dietary sources. In acute toxicity studies, atrazine caused developmental toxicity in the rabbit [no observed effect level (NOEL) 5 mg kg(-1) day(-1)] and cardiotoxicity in a dog chronic study (NOEL 0.5 mg kg(-1) day(-1)); cancer (mammary glands) resulted from lifetime exposure. The mammary tumors, which occurred specifically in female Sprague-Dawley rats, were malignant, increased in a dose-dependent manner and were also observed with other, related triazines. Evidence for a genotoxic basis for these tumors was either equivocal or negative. Triazines have been shown to be clastogenic in Chinese hamster ovary cells, in vitro, but without showing a convincing dose/response relationship. Atrazine can be converted into genotoxic N-nitrosoatrazine in the environment or the digestive system, suggesting that N-nitrosamines derived from triazines could be oncogenic. However, it was concluded that N-nitrosotriazines are unlikely to play a significant role in triazine-induced rat mammary gland tumors. An endocrine basis for the mammary tumors, involving premature aging of the female SD rat reproductive system, has been proposed. A suppression of the luteinizing hormone surge during the estrus cycle by atrazine leads to the maintenance of elevated blood levels of 17beta-estradiol (E2) and prolactin. The mechanism for tumor development may include one or more of the following: the induction of aromatase (CYP19) and/or other P450 oxygenases, an antagonist action at the estrogen feedback receptor in the hypothalamus, an agonist action at the mammary gland estrogen receptor or an effect on adrenergic neurons in the hypothalamic-pituitary pathway. None of these has been excluded as a target because there has been a lack of a rigorous attempt to address the mechanism of action for mammary tumors at the molecular level. The potential occupational exposure to atrazine was assessed during mixing, loading and application. Absorbed daily dosage values were 1.8-6.1 microg kg(-1) day(-1). The MOS values (animal NOEL/human exposure) for short-term (acute) exposure were 820-2800. Longer-term occupational exposure and risk were also calculated. Detectable crop residues are generally absent at harvest. Theoretical calculations of acute dietary exposure used tolerance levels, along with secondary residues, and water, for which there is a maximum contamination level; atrazine plus the three main chlorotriazine metabolites were combined. MOS values were above 2000 for all population subgroups. Dietary exposure to atrazine is therefore extremely unlikely to result in human health hazard. Recent publications have reported a possible feminization of frogs, measured in laboratory and field studies. This is assumed to be due to the induction of aromatase, but no measurements of enzyme activity have been reported. In field studies, the water bodies with the greatest numbers of deformed frogs sometimes had the lowest concentrations of atrazine. Other studies have also cast doubt on the feminization theory, except perhaps at very high levels of atrazine. Epidemiology studies have investigated the possibility that atrazine may result in adverse effects in humans. Although some studies have claimed that atrazine exposure results in an elevated risk of prostate cancer, the published literature is inconclusive with respect to cancer incidence.     

Preliminary validation of a point‐of‐care ethylene glycol test for cats

Objective – To evaluate the sensitivity and specificity of a newly available, semi‐quantitative, cage‐side test for the detection of ethylene glycol (EG) toxicosis in cats. Design – Prospective, laboratory study. Setting – University teaching hospital. Animals – This study utilized samples from 57 cats, whose blood had been anticoagulated with EDTA and submitted to the hospital's laboratory for a complete blood count. Samples were centrifuged, and the plasma separated, aliquoted, and immediately frozen at ?30 °C. Interventions – Samples were randomly divided into 2 primary groups (Group 1: no EG added, Group 2: EG added). Twenty microliters of plasma from each of the Group 1 samples was applied directly to the test strip. Plasma samples from Group 2 had EG added at different concentrations to achieve approximate final concentrations of 20, 60, or 80 mg/dL. These samples were then applied to the test strip. Measurements – Two readers who were blinded to the sample preparation procedure and isolated from each other were asked to categorically interpret the colorimetric reaction on the randomly presented test strips. Main Results – The agreement of the 2 reviewers at the 3 different levels of EG concentrations (20, 60, 80 mg/dL) were 0.7, 0.7, and 0.5, respectively. Thus, the readers demonstrated substantial agreement while reading the 2 lower concentrations, while at 80 mg/dL the level of agreement was moderate. Overall, the sensitivity of the assay increased as the concentration of EG increased (reviewer 1: 67%, 67%, 86%; reviewer 2: 56%, 89%, 100%), while the specificity of the assay decreased with increasing concentrations of EG (reviewer 1: 77%, 45%, 50%; reviewer 2: 77%, 53%, 25%). Conclusions: Because of the likelihood for false negatives and false positives, results from this test must be viewed in light of clinical data and should not be relied upon as a lone diagnostic test.     

土壤中呋喃丹杀虫双p'p-DDT对蚯蚓的单一及复合毒性的研究

采用江苏省常熟农业生态试验站土壤,均匀混入农药后,调节适当的土壤水分,在人工气候箱内保持温度为20℃、湿度为80%的条件下,分别在第2、4、7、10、15d观察蚯蚓(赤子爱胜蚓Eiseniafoetida)的急性中毒状态及死亡情况并进行数据分析。结果表明,蚯蚓在含有单一杀虫双和呋喃丹的土壤中,分别表现出不同的中毒状态和毒性效应,呋喃丹大于杀虫双对蚯蚓的毒性。在10d时杀虫双及呋喃丹的LC50分别为227.57mg·kg-1和28.28mg·kg-1。当土壤中杀虫双和呋喃丹2种农药复合存在时,蚯蚓主要表现为呋喃丹的中毒状态,2种农药复合后对蚯蚓的致死毒性有显著的影响。本文还研究了p'p-DDT作为背景时,2种农药分别对蚯蚓的复合毒性。结果发现,当土壤中p'p-DDT的含量为10mg·kg-1时,无论是单一还是复合对蚯蚓的毒性基本上没有表现出明显的影响。     

农药对家蚕的毒性及安全性评价研究进展

家蚕是农业生态系统中对农药十分敏感的重要经济昆虫,又是非靶标生物的代表物种之一。本文综述了农药对家蚕的毒性及安全性评价的研究现状和进展。     

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat,Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg^-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min^-1 mg^-1 cytosolic protein, respectively). © 1997 SCI.