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Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

Purpose: To characterize the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics of paclitaxel in patients with abnormal liver function., Patients and Methods: Adults with tumors appropriate for paclitaxel therapy who had abnormal liver function tests were eligible. Patients were assigned to one of three treatment cohorts: I, AST level twofold normal and bilirubin level less than 1.5 mg/dL; II, bilirubin level 1.6 to 3.0 mg/dL; and III, bilirubin level greater than 3.0 mg/dL. Doses were explored in at least three patients within each cohort. Although designed to assess a 24-hour infusion schedule, the trial was extended to also assess a 3-hour regimen. Pharmacokinetics were to be studied in all patients., Results: Eighty-one patients were assessable for toxicity. Patients with bilirubin levels greater than 1.5 mg/dL had substantial toxicity at all doses explored, whereas the toxicity for patients with elevated AST levels occurred at doses that ranged from 50 to 175 mg/m2 administered over 24 hours. In most patients, the DLT was myelosuppression. The pharmacokinetic data were insufficient to adequately evaluate the relationship between pharmacokinetics and toxicity in patients who received 24-hour infusions but provided evidence of a longer exposure to paclitaxel than anticipated for the doses used in this study in the 3-hour infusion group., Conclusion: If paclitaxel is used for patients with elevated levels of AST or bilirubin, dose reductions are necessary, and an increase in toxicity can be anticipated. The increased myelosuppression observed is at least partially because of altered paclitaxel pharmacokinetics in such patients.

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3' nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambiquan patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations.

The neoplastic transformation of cultured rat liver epithelial cells by various means has consistently been associated with the development of resistance to the mito-inhibitory effect of transforming growth factor beta (TGF-beta), suggesting that such phenotype plays a mechanistic role during the transformation of these cells. We have studied the induction of the "TGF-beta-resistant" phenotype in a clonal strain of early passage WB-F344 normal cultured rat liver epithelial cells, the proliferation of which was markedly inhibited by TGF-beta. The control WB cells in continuous culture slowly developed TGF-beta resistance. However, when the same cells were exposed to step-wise increases of TGF-beta concentration in their culture medium, the development of TGF-beta resistance was accelerated. Cells which had been grown in medium containing 1 ng/ml TGF-beta developed colony-forming capacity in soft agar containing epidermal growth factor. Cells which were grown in media containing 5 and 10 ng/ml TGF-beta demonstrated a low level of colony-forming efficiency in soft agar medium without added epidermal growth factor and tumorigenicity in isogeneic rats. These TGF-beta-resistant cells also exhibited progressively increasing levels of expression of the c-fos and and myc mRNA, and increased resistance to the cytotoxicity of Adriamycin and melphalan. The latter phenomenon was accompanied by an increase in the mdr-1 mRNA expression, cellular glutathione level, and glutathione S-transferase activity. The results suggest that chronic exposure to high concentration of TGF-beta promotes the spontaneous neoplastic transformation of cultured rat liver epithelial cells, and that this process may represent one of the mechanisms of cellular adaptation for induction of the multidrug-resistant phenotype during the carcinogenesis of epithelial cells.

Cultured rat liver epithelial cells (RLE) transformed with repeated treatments of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) demonstrate many features of the common biochemical phenotype of multidrug resistance (MDR) seen in vivo in 'resistant hepatocytes'. The cells have increased glutathione-S-transferase placental subunit (GST-Yp), gamma-glutamyltranspeptidase (GGT), glutathione (GSH) and glutathione peroxidase and are resistant to MNNG. Phenotypically identical RLE cells spontaneously transformed by selective culture conditions showed low levels of GGT and GST and were not resistant to MNNG. Both chemical and spontaneous transformants are cross resistant to doxorubicin although resistance is consistently greater in chemical transformants. No direct correlation was found between the degree of resistance to doxorubicin and MDR gene expression in either of the chemically or spontaneously transformed RLE cells. These observations suggest that in chemical carcinogenesis, other mechanisms of drug detoxification are involved and that MDR expression is not a consistent feature.

A specific biochemical phenotype is expressed during chemical hepatocarcinogenesis, which includes increased activity of the various isoenzyme forms of glutathione S-transferase (GST) composed of class alpha (Ya/Yc), class mu (Yb) and class pi (Yp) gene products. In vitro cell lines of normal and chemically transformed rat liver epithelial cells provide an opportunity to examine the regulation of expression of GST isoenzymes. We have studied the effect of the state of proliferation in culture on both the enzymic activity and the isoenzyme-specific mRNA expression. In normal rat liver epithelial cells (WB-F344), basal expression of the Yp subunit decreases, and of the Yb subunit increases, in cells at confluence compared with those in logarithmic-phase growth. In a subline of WB-F344 cells that has been chemically treated in vitro (GN6), there was greater Yp expression; however, the effect of growth status on both subunits was the same as in the nontransformed WB-344 cells, and the Ya subunit was not expressed. Inhibition of RNA synthesis with actinomycin D was limited, demonstrating pronged half-lives of the GST mRNAs, and shows a slightly greater decrease in GST-Yp specific mRNA levels in the confluent cells. Also nuclear run-off experiments demonstrate identical transcription rate in confluent and pre-confluent cells. These data suggest that the increase in Yp steady-state RNA in preconfluent cells is due to increased stability of the GST-Yp mRNA. In tumor cells derived from GN6 cells, the regulatory effects of growth status on the Yb and Yp expressions were absent. However, in these cells Ya subunit was expressed and this was subject to the effects of cell proliferation. We conclude that the growth status of cells in culture exerts a significant regulatory control on the GST isoenzyme gene expression. The effects are probably mediated at independent regulatory sites in each gene. The state of transformation of rat liver epithelial cells may determine responsiveness to this effect.

In rat chemical hepatocarcinogenesis models, the hepatocytes in the preneoplastic/neoplastic nodules characteristically demonstrate common biochemical changes including significant and often marked elevation in the cellular glutathione (GSH) content and in the activities of the enzymes gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST). Such consistent and concomitant biochemical changes may signify a common regulatory mechanism in the expression of these enzymes. We have utilized a panel of clonally derived rat liver epithelial cell lines that express varying activities of GGT to study the quantitative correlation between these three cellular components of the phase II drug metabolizing enzyme system. The results indicate that in confluent cultures, cells with high GGT activities have significantly higher cellular GSH content, and a linear correlation exists between the glutathione content and the logarithm of the GGT activity. In contrast, the basal activities of GST and GGT were not coordinately regulated. However, most of the chemical carcinogen-treated cell lines, regardless of their GGT activity, expressed higher GST activity than the normal parental rat liver epithelial cells. The basal expressions of both the Yb and Yp subunits of GST were also not correlated with the relative expression of GGT. Since GGT may play an important role in supplying the cells with the basic constituents for the synthesis of GSH and since GSH is an important cellular molecule in the protection of cells from toxic electrophiles, enhancement of GGT activity in preneoplastic/neoplastic nodules of chemical carcinogen-treated rats may represent a necessary biochemical adaptation for the induction of the "resistant" phenotype of these hepatocytes.

The induction of activity of gamma-glutamyl transpeptidase (GGT) by all-trans retinoic acid in a chemically transformed rat liver epithelial cell line has been investigated. Retinoic acid increased the level of the GGT mRNA and this enhancement was progressive, depending on the duration of exposure and on the concentration of retinoic acid in the culture medium. When retinoic acid was removed from cultures which had been exposed to it for 4 days, the induced GGT activity remained unchanged. In contrast to transformed cell lines, retinoic acid did not induce the activity of GGT in normal/non-transformed rat liver epithelial cells.

Uniform hepatic density alterations may be due to fatty infiltration or hemochromatosis. Whole liver density pattern changes may occur with rapidity where there is liver fat accumulation or mobilization. Averaged hepatic densities from concomitantly increased fat and iron accumulation can result in a false impression of liver normalcy. Two cases are presented to illustrate these points and the importance of understanding the patterns in terms of pathophysiology and response to treatment.