Effect of nicotinamide on hepatic microsomal drug metabolising system in tumour-bearing rats and mice

Administration of nicotinamide to Wistar rats (100 mg/kg body wt.) bearing Yoshida sarcoma (ascites) tumour as well as to Swiss and CBA mice (250 mg/kg body wt.) bearing the transplantable fibrosarcoma and the spontaneously induced mammary carcinoma, respectively, was shown to bring about a reversal of the decreased activity of NADPH-cytochrome c reductase in host livers of the tumour-bearing animals. Nicotinamide injection was also shown to bring about a significant increase in the levels of host hepatic cytochromes P-450 and b5 and in the activities of aminopyrine demethylase and UDP-glucuronosyl transferase which were shown to be low in the tumour-bearing rats. Treatment with nicotinamide was shown to be equally effective in reversing the inhibited activities of hepatic drug metabolising enzymes observed in healthy adult rats injected with serum from the tumour-bearing rats. Administration of nicotinamide to adult male rats brought about an increased incorporation of [14C]leucine in hepatic microsomal proteins which was insensitive to inhibition by actinomycin D at a dose of 1 mg/kg body wt. but could be inhibited by the antibiotic at a lower dose of 0.1 mg/kg body wt. as well as by cycloheximide.     

A species/strain comparison of hepatic natural lymphocytotoxic activities in rats and mice

The natural killer (NK) and natural cytotoxic (NC) cell activities in livers from certain rat and mouse strains were compared. This included the two rodent strains used in animal carcinogenicity bioassays, i.e. Fischer 344 and B6C3F1 mice. Sprague-Dawley and Fischer 344 rats exhibited high hepatic NK activity, which was greater than the levels seen in all of the five mouse strains studied. However, the hepatic NC activity in rats was comparable to the activities observed in C57BL and BALB/c mice. An inverse relationship was observed between the two tumoricidal activities in all but one of the mouse strains examined; that is (at 8 weeks of age), NK activity: C3H greater than B6C3F1 greater than CBA greater than BALB/c; NC activity: BALB/c much greater than CBA greater than B6C3F1 greater than C3H. The C57BL mouse strain was the only strain to express both activities at comparatively high levels. Female mice exhibited a similar profile of cytotoxic activities. Rats also possessed high activities of a presently ill-defined tumoricidal activity, this being the spontaneous P815 mastocytoma killing by unstimulated effector cells, over an 18 h period. Both adherent and nonadherent effector cells from rat livers, but only the nonadherent cell population isolated from male mouse livers, exhibited this activity which may represent a distinct hepato-specific population of natural lymphocytotoxic effector cells. The tumoricidal activities in liver-derived cells were greater than those of effector cells isolated from the spleen. The differences in natural immunity reported in this study may be related to the varying background incidences of hepatic tumors, i.e. the mouse strains susceptible to high background incidences of liver tumors have relatively low natural immunity, whereas the two mouse strains resistant to hepatic tumors possess high levels of at least one hepatic NLC activity. Similarly, rats have relatively low hepatic tumor rates and high levels of hepatic natural immunity.     

Molecular basis for the sex-related difference in renal N-nitrosodimethylamine demethylase in C3H/HeJ mice

Previous work with rat and rabbit liver enzymes has demonstrated that cytochrome P450IIE1 is responsible for the metabolism of N-nitrosodimethylamine (NDMA), a widely occurring carcinogen. The present study demonstrated that a similar enzyme also exists in the mouse kidney and is regulated by testosterone. These results can account for the reported sex-related difference in the renal metabolism of NDMA in mouse strains such as C3H/HeJ. NDMA demethylase activities (expressed as pmol/min/mg protein) in kidney microsomes of female and male C3H/HeJ mice were 3.0 +/- 0.7 and 51.9 +/- 11.2, respectively. After testosterone treatment (500 mg/kg b.w. in olive oil, s.c.) for 2 days, the renal NDMA demethylase activity of the female mice was elevated 17-fold. The difference and change in NDMA demethylase activity were accompanied by corresponding differences and changes in P450IIE1 as quantified by immunoblot analysis (using antibodies prepared against rat P450IIE1) as well as in the mRNA level for P450IIE1 as determined by Northern and slot blot analyses (using a cDNA probe containing the coding sequence of rat P450IIE1 gene). Based on gel electrophoresis, the molecular weight of mouse renal P450IIE1 was 52,000 and the size of mouse renal P450IIE1 mRNA was approximately 1.8 kilobases; both were similar to those found in rat liver and kidney. Renal P450IIE1 mRNA levels in female, male, and testosterone-treated female mice were at a ratio of 1:22:20. On the other hand, this testosterone-related difference was not observed in hepatic P450IIE1. In liver microsomes, there were no significant differences in NDMA demethylase activity, P450IIE1 content, and P450IIE1 mRNA level between male and female mice or between untreated and testosterone-treated female mice. The apparent Km value of NDMA demethylase in mouse kidney microsomes (22 to 27 microM NDMA) were similar to that in rat liver microsomes. Renal NDMA demethylase activity was inhibited by a monoclonal antibody prepared against rat P450IIE1. These results suggest that mouse renal P450IIE1 is similar to rat P450IIE1 and is responsible for the low Km form of NDMA demethylase activity. Nevertheless, only the mouse renal enzyme is regulated by testosterone.     

Hepatocarcinogenicity of hexachlorobenzene in rats and the sex difference in hepatic iron status and development of porphyria

Hexachlorobenzene (HCB) was fed to male and female F344 rats as 0.02% of the diet for 15 weeks. Females developed a massive porphyria, due to depression of uroporphyrinogen decarboxylase activity, whereas males did not. Although hepatic non-haem iron levels in control females were 3-5 times greater than males (iron is implicated in the pathogenesis of this condition) preloading the latter with iron did not increase their susceptibility. After 90 weeks of HCB treatment 100% of surviving females had multiple liver tumours which were strongly gamma-glutamyl transpeptidase (GGT) positive and histologically classified as neoplastic nodules or hepatocellular carcinomas. In contrast, only 16% of males developed tumours which were smaller and fewer in number per liver than those in females. Accumulation of porphyrins was still significantly less in males than females although no uroporphyrinogen decarboxylase activity was detected in treated livers of either sex. No differences in porphyrin levels or enzyme activity were found between tumours and surrounding tissue showing that tumours did not revert to a non-porphyric state. The sex difference in tumour response could not be explained by differences in hepatic HCB concentrations. Non-haem iron concentrations of livers fell after HCB treatment for 90 weeks in both sexes and were even lower in tumours. These studies demonstrate that not only are female rats far more sensitive than males to the porphyrinogenic effects of HCB but also to the hepatocarcinogenic actions, suggesting a link between these two manifestations of toxicity that may also apply to other polyhalogenated aromatics.     

The effect of the dose of diethylnitrosamine on the initiation of altered hepatic foci in neonatal female rats

The dose—response characteristics of initiation of hepatocarcinogenesisby diethylnitrosamine (DEN) was investigated in the neonatalfemale rat by means of the quantitative stereologic estimationof altered hepatic foci (AHF) expressing multiple markers. At5 days of age, female Sprague—Dawley rats were given asingle i.p. dose of DEN (0.1–30 mg/kg body wt) or thevehicle (trioctanoin). The semisynthetic AIN-76A diet was providedto half of the rats in each treatment group, while the remainderreceived this diet containing 500 mg phenobarbital (PB)/kg for8 months from weaning until the animals were killed. To ascertainmore exactly the dose—response relationship for initiationby DEN, the number, volume percentage and phenotypes of theresulting AHF were determined by quantitative stereologicalanalysis on serial sections of frozen tissue, each stained forone of four markers of preneoplasia. A linear relationship wasobserved between the dose of DEN (0–30 mg/kg) and thenumber and volume percentage of AHF detected, with each singlemarker or the total number of AHF detected when the placentalisozyme of glutathione S transferase,     

The value of hepatic ultrasound and biochemical liver tests in screening for liver metastases

The records of 92 patients with a known diagnosis of extrahepatic cancer who had undergone hepatic ultrasound, biochemical liver tests (alkaline phosphatase, SGOT, lactic dehydrogenase, and bilirubin levels), and subsequent liver biopsy or autopsy within a 6-week period were reviewed. The sensitivity, specificity, and accuracy of the ultrasound and biochemical tests in the detection of metastatic liver disease were calculated. Although there was no significant difference in the sensitivity of either examination, the ultrasound demonstrated higher specificity and accuracy than the biochemical liver tests. The high sensitivity of hepatic ultrasound prevailed even in patients with normal biochemical liver tests. The sensitivity of hepatic ultrasound was significantly lower in patients with lymphoma compared with patients with colorectal cancer (50% v 100%, P less than .05). Notable incidental extrahepatic findings were reported in 25% of the ultrasound examinations. In institutions skilled in sonography, hepatic ultrasound may be a superior tool in the detection of liver metastases in most solid tumors, excluding lymphoma, and offers the additional advantage of simultaneous biliary tract and perihepatic visualization.     

Morphological and biochemical effects of 1,2-dimethylhydrazine and 1-methylhydrazine in rats and mice

Single toxic doses of 1,2-dimethylhydrazine induced mild centrilobular necrosis of the liver in rats and mice. Ultrastructural studies showed hepatic nuclear changes including nucleolar microsegregation and changes in the endoplasmic reticulum and mitochondria. 1-Methylhydrazine caused little morphological change in the liver. Tumours of the colon and kidney and also massive cystic hyperplasia of the liver were found in some of the rats and tumours of the anal margin and kidney in some of the mice, following single doses of 1,2-dimethylhydrazine. Incorporation of amino acids into rat liver proteins was inhibited by 1,2-dimethylhydrazine, which also caused disaggregation of hepatic polysomes. No effects on hepatic protein synthesis by 1,1-dimethylhydrazine or 1-methylhydrazine were observed. Similarities between the effects of 1,2-dimethylhydrazine, cycasin and dimethylnitrosamine are discussed.     

An inverse correlation between hepatic ornithine decarboxylase and S-adenosylmethionine in rats

The comparative effects of the subchronic administration to rats of ethionine-supplemented and of chemically defined methyl-deficient diets on the hepatic levels of S-adenosylmethionine (SAM) and of ornithine decarboxylase (ODC), an enzyme marker of cell proliferation, were studied. Both treatments led to decreased hepatic levels of SAM and to marked increased activities in ODC. Both systems led to significant inverse correlations between ODC and SAM. In rats fed the methyl-deficient diets, hepatic levels of SAM were generally proportional to the dietary content of methionine and choline. The metabolic increases in S-adenosylmethionine decarboxylase (SAMDC) observed in the livers of methyl-deficient rats were proportional to the changes seen in ODC.     

Induction of hepatic aneuploidy in vivo by tamoxifen, toremifene and idoxifene in female Sprague-Dawley rats

Since tamoxifen is efficacious for the prevention of secondprimary breast neoplasms in humans and has a low reported incidenceof acute side effects, several structurally related compoundshave been developed for the treatment of breast cancer includingtoremifene and idoxifene. We have compared the karyotypic alterationsthat occur after a single per os administration of 35 mg/kgof tamoxifen, toremifene or idoxifene to female Sprague-Dawleyrats. One day following treatment, the rats were sacrificedand the hepatocytes isolated and cultured. After 47 h in culture,colcemid was added for 3 h prior to harvest of the hepatocytesfor karyotypic evaluation. At least 100 metaphase spreads wereexamined for each of five rats per treatment. Toremifene resultedin aneuploidy in 50±7% of the cells examined and idoxifeneinduced a 57±4% aneuploidy compared with the 85±7%level induced by tamoxifen. Since the level of aneuploidy insolvent-treated rats was 3±3%, the induction of aneuploidyin at least 50% of the cells from rats treated with tamoxifen,toremifene or idoxifene was highly significant Analysis of electronmicrographs of cultures treated with these antiestrogens demonstrateda range of phenotypes including multipolar spindles in toremifene-treatedrats and condensed chromosomes in the presence of an intactnuclear envelope in occasional idoxifene-treated rat hepatocytes.The exclusion of chromosomes from the spindle apparatus andthe lagging of some chromosomes on the metaphase plate correlatewith the high rate of induction of aneuploidy in the rat liveras determined by karyotypic analysis of hepatocytes from ratstreated with these triphenylethylenes.     

Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H2O2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.     

Change in the gene expression of hepatic tamoxifen-metabolizing enzymes during the process of tamoxifen-induced hepatocarcinogenesis in female rats

Altered gene expression of the enzymes responsible for tamoxifen metabolism during the process of tamoxifen-induced hepatocarcinogenesis in female Sprague-Dawley rats was examined by the RT-PCR method. Treatment of rats with tamoxifen (20 mg/kg body/day) for 52 weeks, but not the 1 day, 2 or 12 week treatments, resulted in the formation of the liver hyperplastic nodules. The gene expression of CYP3A subfamily enzymes, especially CYP3A1, responsible for not only detoxification (N-demethylation) but also activation (alpha-hydroxylation) of tamoxifen, was increased by the tamoxifen treatments for 2 and 12 weeks, whereas after the 52 week treatment, the expression in the induced nodules returned to the control level. The gene expression of SULT2A subfamily sulfotransferases, especially HSTa, responsible for metabolic activation of alpha-hydroxytamoxifen was decreased to a level <20% of the control in the nodules, although no significant change in the expression was observed in the liver of rats treated with tamoxifen for 1 day, 2 or 12 weeks. On the other hand, the gene expression of CYP3A2 and flavin-containing monooxygenase 1 (FMO1), responsible for the N-demethylation and N-oxidation, respectively, of tamoxifen was increased in a time-dependent fashion up to the 52 week treatment. Although the gene expression of UDP-glucuronosyltransferase(s), which might be responsible for detoxification of tamoxifen, was also increased by the tamoxifen treatment for 2 or 12 weeks, it decreased to the control level in the nodules after the 52 week treatment. The present findings demonstrate that in the early stage of the formation of the liver hyperplastic nodules by tamoxifen, the genes of the enzymes responsible for not only detoxification but also activation of tamoxifen were activated, whereas in the later stage (in the nodules), the genes of the detoxification enzymes, CYP3A2 and FMO1, remained active, but those of the activation enzymes such as CYP3A1 and HSTa were suppressed.     

Differences in metabolism of vinylidene chloride between mice and rats

The present finding that mice metabolize a greater proportion of an oral dose (50 mg/kg) of vinylidence chloride. (1,1 - dichloroethylene, DCE) than rats implies (a) that the efficiency of DCE metabolism follows the known activity of cytochrome P-450 in the organs of these animals, and (b) that, in accordance with the LD₅₀ values, the real exposure (expressed as the amount of DCE metabolized) is relatively higher for orally dosed mice than rats, and (c) that DCE carcinogenicity would appear to be more likely in mice than rats.     

Organ specificity in the microsomal activation and toxicity of N-nitrosomethylbenzylamine in various species

The microsomal metabolism of the rat esophageal carcinogen N-nitrosomethylbenzylamine (NMBZA) at the methylene carbon atom to yield benzaldehyde was studied in various organs of a number of species to determine the role of metabolic activation in the carcinogenicity or toxicity of the nitrosamine. In the Sprague-Dawley rat, NMBZA was metabolized by microsomes from liver, lung, and esophageal mucosa. In the F344 rat and rabbit, metabolic activity was present in both liver and esophageal mucosa, the only tissues studied in these species. In contrast, in the Syrian hamster and BALB/cByJ mouse, NMBZA debenzylation was undetectable in the esophagus but occurred at relatively high rates in liver, lung, and kidney. The forestomach mucosa exhibited undetectable levels of activity in the Sprague-Dawley rat and BALB/cByJ mouse, although in the hamster, it was present at a very low level. Administration of a dose of NMBZA acutely toxic to the rat (18 mg/kg i.p.) resulted in significant cellular damage only to the rat esophageal mucosa, no other tissues examined in the rat, hamster, or mouse being affected. These observations, together with the available data on carcinogenicity of the nitrosamine in the rat and rabbit, suggest that in the esophagus, at least, metabolic activation of NMBZA is necessary to elicit its toxic and/or carcinogenic effect. However, NMBZA is also metabolized at a high rate in the liver of all species but is not toxic or carcinogenic in this tissue, suggesting that other factors besides metabolic activation must be involved in the resistance of hepatocytes to the effects of the nitrosamine. Microsomes prepared from human esophageal mucosa from six patients metabolized NMBZA at rates that were either undetectable or approximately 70 times lower than in the Sprague-Dawley rat.     

肿瘤细胞在活体小鼠肝脏微循环中动态变化的可视性研究

目的:在小鼠活体内动态观察肿瘤细胞在肝脏微循环中的运动、分布及凋亡。方法:用Calcein AM荧光标记B16F1黑色素瘤细胞,将其注射入C57BL/6小鼠的肠系膜静脉,在荧光倒置显微镜下动态观察肿瘤细胞在肝脏微循环中的运动及分布,测量并计算肿瘤细胞在终末门静脉中的运动速度。注射肿瘤细胞8小时后,切取肝脏,用凋亡标记荧光素对肝组织切片进行原位DNA末端标记,在荧光显微镜下计算凋亡细胞百分比。结果:B16F1黑色素瘤细胞进入肝脏微循环中后,90%的细胞通过机械性嵌顿停留在肝窦中,10%的肿瘤细胞通过黏附停留在终末门静脉中,B16F1细胞在肝脏终末门静脉中的运动速度为648±53μm/s。在注射肿瘤细胞8小时后,在肝窦中约有30%的肿瘤细胞发生凋亡,在终末门静脉中约有10%的肿瘤细胞发生凋亡。结论:利用荧光标记的肿瘤细胞及荧光倒置显微镜为探讨肿瘤肝转移机制提供了一个良好的活体观察方法。     

The effect of the format of administration and the total dose of phenobarbital on altered hepatic foci following initiation in female rats with diethylnitrosamine

The effect of changing the format of administration as well as the total dose of the promoting agent phenobarbital (PB) on the development of altered hepatic foci (AHF) was determined in an initiation-promotion protocol with female rats fed the purified AIN-76 diet. Effects on the total number of AHF and the volume percentage of liver occupied by AHF were determined for four histochemical markers, the placental form of glutathione S-transferase, gamma-glutamyl-transpeptidase canalicular ATPase, and glucose-6-phosphatase after 16 and 60 weeks of promotion with varying doses and formats of PB, as well as for a further 16-week period in which no PB was administered. At the 16-week point, animals fed 0.1% PB continuously exhibited the largest number and volume percentage of AHF, whereas rats fed 0.1% PB for 4 days followed by 10 days of no PB with continuous repetition of this pattern during the 16-week treatment period exhibited no increase in the number of AHF over control and only a slight increase in volume percentage. Rats fed a continuous repetition of 0.2% PB for 2 days followed by 12 days of no PB exhibited an intermediate increase in the number of AHF as well as the volume percentage fraction after 16 weeks of this regimen. After 60 weeks of feeding PB by these three different formats, the numbers of AHF observed in these groups were equivalent and had increased above those seen after 16 weeks of feeding. The volume percentage occupied by the AHF in these three groups was also similar, although animals receiving 0.2% PB intermittently showed a significantly lower volume percentage than animals receiving 0.1% PB continuously for 60 weeks. When animals were maintained for an additional 16 weeks without PB feeding, the numbers of AHF decreased dramatically, much more so in animals fed PB intermittently, whereas the volume percentage fraction of AHF in livers of animals receiving 0.1% PB continuously for 60 weeks almost doubled. In contrast, the volume percentage fraction of AHF in livers of animals receiving PB intermittently for 60 weeks followed by 16 weeks of no PB was slightly less. Examination of the individual size classes of AHF showed little change in their distribution at 16 and 60 weeks, but after 16 weeks of PB withdrawal (76 weeks total time), the distribution of AHF in animals that had received 0.1% PB continuously for 60 weeks exhibited a decidedly greater shift to larger AHF than animals receiving PB intermittently for the 60-week period.(ABSTRACT TRUNCATED AT 400 WORDS)