Toxic Rep Ser. 2000 Feb;46:1-C7.
Methapyrilene hydrochloride is a histamine H(1)-receptor antagonist that was an active ingredient in many over-the-counter cold and allergy medications. In the mid- to late 1970s, studies in rats suggested that methapyrilene hydrochloride was a hepatocarcinogen, and the drug was removed from these preparations. In most cases, methapyrilene hydrochloride was replaced by pyrilamine maleate, a structurally similar analogue. As part of a program to investigate mechanisms of toxicity whereby structurally similar chemicals produce different toxicities, these chemicals were studied for induction of cell proliferation and protein alterations by two-dimensional gel electrophoresis in the liver of F344/N rats. A complete toxicologic evaluation was not needed for this research-oriented study. Rather, the goal of the present study was to provide retrospective data from subchronic toxicity studies with the known rat carcinogen methapyriline hydrochloride that could then be used to predict the potential carcinogenicity of unknown chemical agents and that could also be compared with similar data on the structural analogue pyrilamine maleate. Pyrilamine maleate differs from methapyrilene hydrochloride in the substitution of the thienyl ring with a paramethoxyphenyl ring. Pyrilamine maleate has been shown to produce an equivocal increase in the incidences of liver neoplasms in rats in 2-year feed studies, but only at 2,000 ppm, indicating that its potency, if any, to produce neoplasms is much less than that of methapyriline hydrochloride. The hepatocarcinogenic peroxisome proliferator Wy-14,643 was included in this study as a positive control that is known to induce cell proliferation, as well as protein alterations, in the liver. In the 14-week study of methapyrilene hydrochloride, groups of 40 male F344/N rats were given 0, 50, 100, 250, or 1,000 ppm methapyrilene hydrochloride, 1,000 ppm pyrilamine maleate (negative control), or 50 ppm Wy-14,643 ( positive control) in feed. Rats in all groups were administered bromodeoxyuridine (BrdU) by osmotic minipump for the assessment of hepatocyte proliferation. Ten rats from each group were evaluated on days 15, 29, and 43 and at 14 weeks. At these times, samples of liver tissue were analyzed for evidence of cell proliferation via BrdU labeling and proliferating cell nuclear antigen (PCNA) labeling. There were no exposure-related deaths. Low mean body weights were generally observed in the 1,000 ppm methapyriline hydrochloride group and in the positive control group. Final mean body weights and mean body weight gains of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group at all time points. The final mean body weights of rats in the positive control group were significantly less than those of the untreated control group for rats evaluated on days 29 and 43 and at week 14; the mean body weight gains of rats in the positive control group were significantly less than those of the untreated control group on day 29 and at week 14. Feed consumption by rats exposed to 1,000 ppm methapyrilene hydrochloride was significantly less than that by the untreated control group throughout the study. The predominant clinical observation related to methapyrilene hydrochloride exposure was thinness in rats exposed to 1,000 ppm; this finding was first observed on day 29. On days 29 and 43 and at 14 weeks, the absolute liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group. At all time points, the relative liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride and the absolute and relative liver weights of positive control rats were significantly greater than those of the untreated control group. No significant differences in liver weights were observed between the negative and untreated control groups at any time point. Hepatic lesions were observed predominantly in the 250 and 1,000 ppm methapyrilene hydrochloride groups and in the positive control group. The incidences of bile duct hyperplasia, hepatocyte necrosis, hepatocyte mitosis, and hepatocyte hypertrophy in rats in the 1,000 ppm group were significantly greater than those in the untreated control group at all time points. The severities of hepatocyte hypertrophy and hepatocyte mitosis in 1,000 ppm rats were generally mild to moderate; the lesions occurring in 250 ppm animals were less severe. At each time point, the incidence of bile duct hyperplasia in 250 ppm rats was significantly greater than that in the untreated control group. The incidences of hepatocyte mitosis on days 15 and 29 and the incidences of hepatocyte necrosis on days 29 and 43 in rats in the 250 ppm group were significantly greater than those in the untreated control group. Incidences of pigmentation in the 250 and 1,000 ppm methapyrilene hydrochloride groups were significantly greater than those in the untreated control group on days 29 and 43 and at 14 weeks. In the positive control group, the incidences of granulomatous inflammation were significantly greater than those in the untreated control group on days 15, 29, and 43. The incidences of hepatocyte hypertrophy and hepatocyte mitosis in the positive control group were significantly greater than those in the untreated control group on days 15, 29, and 43. The incidence of hepatocyte hypertrophy was also significantly increased in the positive control group at 14 weeks. The severity of hepatocyte hypertrophy in the 1,000 ppm methapyrilene hydrochloride group was generally greater than that in the positive control group at each time point. In general, methapyriline hydrochloride produced a dramatic and sustained increase in hepatic cell proliferation over 14 weeks, whereas pyrilamine maleate at the same concentration produced few if any effects. Wy-14,643 also induced a large increase in cell proliferation which declined over time, as has been observed in previous studies. The mean BrdU labeling indexes of the 250 and 1,000 ppm methapyrilene hydrochloride groups were generally significantly greater than those of the untreated controls at all time points. In the negative control group, the BrdU labeling index was significantly less than that of the untreated control group on day 29. The BrdU labeling index in the positive control group was significantly greater than that of the untreated control group at all time points. On day 43 and at week 14, the mean PCNA labeling indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group. The mean PCNA labeling indexes of the negative control group were significantly less than those of the untreated control group on days 29 and 43. On day 29, the mean PCNA labeling index of the positive control group was significantly greater than that of the untreated control group. The mitotic indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group at all time points. The mitotic indexes of the 250 ppm group were significantly greater than those of the untreated control group on day 43 and at week 14. At least 32 proteins underwent significant abundance changes at the highest exposure concentration of methapyrilene hydrochloride, and 39 protein changes were observed in the positive control group. Many, but not all, of the protein changes in the methapyrilene hydrochloride-exposed animals also occurred in the positive control group. Treatment with pyrilamine maleate produced no significant quantitative protein changes, as judged by the same criteria used for methapyrilene hydrochloride and Wy-14,643. Methapyrilene hydrochloride produced covalent modification of mitochondrial proteins as measured by the charge modification index. PCNA abundance in liver samples from the 250 and 1,000 ppm methapyrilene hydrochloride exposure groups on day 43 was significantly greater than that of the untreated control group. Results of tests for induction of mutagenicity by methapyrilene hydrochloride were negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in L5178Y mouse lymphoma cells, with and without S9 metabolic activation. However, positive responses were obtained in cytogenetic tests with cultured Chinese hamster ovary cells, in which methapyrilene hydrochloride induced sister chromatid exchanges and chromosomal aberrations. The increases in sister chromosome exchanges were obtained with and without S9, but chromosomal aberrations were increased only in the presence of S9. In summary, the significance of the increased hepatic cell proliferation and the protein alterations observed in this study is not definite, but may be of predictive value for assessing the toxicity and carcinogenicity of chemicals in preclinical assays. A chemical which does not produce an increase in cell proliferation or a large number of protein changes may be considered safer than a similar chemical that produces many such changes.
盐酸美吡拉敏是一种组胺H(1)受体拮抗剂，曾是许多非处方感冒和过敏药物的有效成分。在20世纪70年代中后期，对大鼠的研究表明盐酸美吡拉敏是一种肝致癌物，该药物随后从这些制剂中被去除。在大多数情况下，盐酸美吡拉敏被马来酸氯苯那敏取代，后者是一种结构相似的类似物。作为一项研究结构相似化学物质产生不同毒性的毒性机制的计划的一部分，通过二维凝胶电泳研究了这些化学物质在F344/N大鼠肝脏中诱导细胞增殖和蛋白质改变的情况。对于这项以研究为导向的研究，不需要进行完整的毒理学评估。相反，本研究的目标是提供来自已知大鼠致癌物盐酸美吡拉敏的亚慢性毒性研究的回顾性数据，这些数据可用于预测未知化学物质的潜在致癌性，也可与结构类似物马来酸氯苯那敏的类似数据进行比较。马来酸氯苯那敏与盐酸美吡拉敏的不同之处在于其噻吩环被对甲氧基苯基环取代。在为期两年的喂养研究中，已证明马来酸氯苯那敏在大鼠肝脏肿瘤发生率上有不明确的增加，但仅在2000 ppm时出现，这表明其产生肿瘤的能力（如果有的话）远低于盐酸美吡拉敏。肝致癌物过氧化物酶体增殖剂Wy-14,643被纳入本研究作为阳性对照，已知其可诱导肝脏中的细胞增殖以及蛋白质改变。在对盐酸美吡拉敏进行的为期14周的研究中，将40只雄性F344/N大鼠分成几组，分别给予含0、50、100、250或1000 ppm盐酸美吡拉敏、1000 ppm马来酸氯苯那敏（阴性对照）或50 ppm Wy-14,643（阳性对照）的饲料。所有组的大鼠通过渗透微型泵给予溴脱氧尿苷（BrdU）以评估肝细胞增殖。在第15、29和43天以及14周时对每组的10只大鼠进行评估。在这些时间点，通过BrdU标记和增殖细胞核抗原（PCNA）标记分析肝组织样本以寻找细胞增殖的证据。没有与暴露相关的死亡。在1000 ppm盐酸美吡拉敏组和阳性对照组中普遍观察到平均体重较低。在所有时间点，暴露于1000 ppm盐酸美吡拉敏的大鼠的最终平均体重和平均体重增加量均显著低于未处理的对照组。在第29天、43天和14周评估时，阳性对照组大鼠的最终平均体重显著低于未处理的对照组；在第29天和14周时，阳性对照组大鼠的平均体重增加量显著低于未处理的对照组。在整个研究过程中，暴露于1000 ppm盐酸美吡拉敏的大鼠的饲料消耗量显著低于未处理的对照组。与盐酸美吡拉敏暴露相关的主要临床观察结果是暴露于1000 ppm的大鼠消瘦；这一发现最早在第29天观察到。在第29天、43天和14周时，暴露于1000 ppm盐酸美吡拉敏的大鼠的绝对肝脏重量显著低于未处理的对照组。在所有时间点，暴露于1000 ppm盐酸美吡拉敏的大鼠的相对肝脏重量以及阳性对照大鼠的绝对和相对肝脏重量均显著高于未处理的对照组。在任何时间点，阴性对照组和未处理的对照组之间的肝脏重量均未观察到显著差异。肝脏病变主要在250和1000 ppm盐酸美吡拉敏组以及阳性对照组中观察到。在所有时间点，1000 ppm组大鼠的胆管增生、肝细胞坏死、肝细胞有丝分裂和肝细胞肥大的发生率均显著高于未处理的对照组。1000 ppm大鼠中肝细胞肥大和肝细胞有丝分裂的严重程度一般为轻度至中度；250 ppm动物中出现的病变较轻。在每个时间点，250 ppm大鼠的胆管增生发生率均显著高于未处理的对照组。250 ppm组大鼠在第15天和29天的肝细胞有丝分裂发生率以及在第29天和43天的肝细胞坏死发生率均显著高于未处理的对照组。在第29天、43天和14周时，250和1000 ppm盐酸美吡拉敏组的色素沉着发生率均显著高于未处理的对照组。在阳性对照组中，在第15天、29天和43天肉芽肿性炎症的发生率均显著高于未处理的对照组。在第15天、29天和43天，阳性对照组中肝细胞肥大和肝细胞有丝分裂的发生率均显著高于未处理的对照组。在14周时，阳性对照组中肝细胞肥大的发生率也显著增加。在每个时间点，1000 ppm盐酸美吡拉敏组中肝细胞肥大的严重程度一般大于阳性对照组。总体而言，盐酸美吡拉敏在14周内使肝细胞增殖显著且持续增加，而相同浓度的马来酸氯苯那敏几乎没有产生任何影响。Wy-14,643也诱导了细胞增殖的大幅增加，且如先前研究中所观察到的，这种增加随时间下降。在所有时间点，250和1000 ppm盐酸美吡拉敏组的平均BrdU标记指数一般均显著高于未处理的对照组。在阴性对照组中，第29天的BrdU标记指数显著低于未处理的对照组。在所有时间点，阳性对照组的BrdU标记指数均显著高于未处理的对照组。在第43天和14周时，1000 ppm盐酸美吡拉敏组的平均PCNA标记指数显著高于未处理的对照组。在第29天和43天，阴性对照组的平均PCNA标记指数显著低于未处理的对照组。在第29天，阳性对照组的平均PCNA标记指数显著高于未处理的对照组。在所有时间点，1000 ppm盐酸美吡拉敏组的有丝分裂指数均显著高于未处理的对照组。在第43天和14周时，250 ppm组的有丝分裂指数显著高于未处理的对照组。在盐酸美吡拉敏的最高暴露浓度下，至少有32种蛋白质的丰度发生了显著变化，在阳性对照组中观察到39种蛋白质变化。在盐酸美吡拉敏暴露动物中发生的许多（但不是全部）蛋白质变化也在阳性对照组中出现。按照用于盐酸美吡拉敏和Wy-14,643的相同标准判断；用马来酸氯苯那敏处理未产生显著的定量蛋白质变化。通过电荷修饰指数测量，盐酸美吡拉敏产生了线粒体蛋白质的共价修饰。在第43天，来自250和1000 ppm盐酸美吡拉敏暴露组的肝脏样本中PCNA的丰度显著高于未处理的对照组。在有和没有S9代谢活化的情况下，盐酸美吡拉敏在鼠伤寒沙门氏菌菌株TA98、TA100、TA1535和TA1537以及L5178Y小鼠淋巴瘤细胞中的致突变性测试结果均为阴性。然而，在培养的中国仓鼠卵巢细胞的细胞遗传学测试中获得了阳性反应，其中盐酸美吡拉敏诱导了姐妹染色单体交换和染色体畸变。无论有无S9，均可观察到姐妹染色体交换增加，但仅在有S9的情况下染色体畸变增加。总之，本研究中观察到的肝细胞增殖增加和蛋白质改变的意义尚不确定，但可能对临床前试验中评估化学物质的毒性和致癌性具有预测价值。一种不引起细胞增殖增加或大量蛋白质变化的化学物质可能被认为比产生许多此类变化的类似化学物质更安全。
