Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens

Williams, Gary M.; Laspia, Michael F.; Dunkel, Virginia C.

doi:10.1016/0165-1161(82)90003-6

Cited by 251 publications

(46 citation statements)

References 14 publications

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“…Primary mouse hepatocytes from ATX and wild-type mice were obtained by liver perfusion (59). In brief, mice were anesthetized and a V incision was made to expose the internal organs.…”

Section: Methodsmentioning

confidence: 99%

Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice

Madden¹,

Finegold

Slagle³

2000

J Virol

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Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human ␣-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.The hepatitis B virus (HBV) X protein (HBx) is a 17-kDa regulatory protein necessary for the establishment of hepadnaviral infection in woodchucks and, presumably, in all mammals (9, 65). Detectable in both the cytoplasm and nucleus of infected cells (13,38,48,56), the essential role(s) for HBx in the life cycle of the virus remains to be established. Although HBx does not bind DNA directly, it is capable of transactivating cellular genes (reviewed in references 6, 8, 45, and 51) and can induce protein kinase signaling cascades (3,13,29,37). In addition to interacting with numerous cellular proteins (reviewed in reference 16), HBx demonstrates a deoxy-ATPase activity (12).Chronic infection with HBV is considered a major risk factor for the development of human hepatocellular carcinoma (HCC) (1, 49), but the exact mechanism(s) by which HBV infection leads to the development of liver cancer remains to be elucidated. An increased rate of hepatocyte cell death and regeneration caused by the host immune response to viral antigens undoubtedly contributes to the pathology of chronic HBV infection (reviewed in references 11 and 49). Several lines of evidence indicate that HBx may also contribute to the development of HCC. Although the majority of HBx transgenic mouse lines do not demonstrate an increased incidence of liver cancer under normal conditions (5,14,21,34,39,43), such mice are more susceptible to the tumorigenic effects of hepatocarcinogens (14, 50) or the oncogene c-myc (54). Based on these observations, HBx is believed to be an important cofactor in HBV-associated liv...

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“…Primary mouse hepatocytes from ATX and wild-type mice were obtained by liver perfusion (59). In brief, mice were anesthetized and a V incision was made to expose the internal organs.…”

Section: Methodsmentioning

confidence: 99%

Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice

Madden¹,

Finegold

Slagle³

2000

J Virol

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“…A decrease in the number of colonies appearing after 48 hr of incubation relative to the negative control is the measure of toxicity. (4,5).…”

Section: Salmonella Mutagenicity Testmentioning

confidence: 99%

Mutagenicity testing of diethylene glycol monobutyl ether.

Thompson¹,

Coppinger²,

Valencia³

et al. 1984

Environ Health Perspect

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The mutagenic potential of diethylene glycol monobutyl ether (diEGBE) was examined with a Tier I battery of in vitro assays followed by a Tier II in vivo Drosophila sex-linked recessive lethal assay. The in vitro battery consisted of: the Salmonella mutagenicity test, the L5178Y mouse lymphoma test, a cytogenetics assay using Chinese hamster ovary cells and the unscheduled DNA synthesis (UDS) assay in rat hepatocytes. Results of the Salmonella mutagenicity test, the cytogenetics test, and the rat hepatocyte assay were negative at concentrations up to 20 FL/plate, 7.92 ,uL/mL, and 4.4 ,uL/mL, respectively. Toxicity was clearly demonstrated at all high doses. A weak, but dose-related increase in the mutation frequency (4-fold increase over the solvent control at 5.6 UL/mL with 12% survival) was obtained in the L5178Y lymphoma test in the absence of metabolic activation. Results of the mouse lymphoma assay were negative in the presence of the S-9 activation system. The significance of the mouse lymphoma assay results were assessed by performing the Tier II sex-linked recessive lethal assay in Drosophila in which the target tissue is maturing germinal cells. Both feeding (11,000 ppm for 3 days) and injection (0.3 ,uL of -14,000 ppm solution) routes of administration were employed in the Drosophila assay. Approximately 11,000 individual crosses with an equal number of negative controls were performed for each route of administration. diEGBE produced no increase in recessive lethals under these conditions. We conclude that the weak response in the mouse lymphoma assay does not have mutagenic significance because of the clear negative obtained in the in vivo assay which detects the same types of mutations in germinal cells.

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“…The widely used Salmonellalmicrosome mutagenicity assay developed and updated by Ames (28) measures back mutations to histidine independence of histidine mutants of Salmonella typhimurium and can be conducted with strains that are also repair-deficient. The assay is highly sensitive (2) but also has the limitation that several chemicals not known to be carcinogenic have been positive (57). The use of a liver S-9 fraction for bioactivation provides an artifactual bioactivation system in which detoxification is not operative.…”

Section: Introductionmentioning

confidence: 99%

“…Both methapyrilene (12) and tamoxifen (10) (52,57). The best established are measurement of DNA binding of radiolabeled chemical (27) and the 32P-postlabeling technique for DNA adducts (39).…”

Section: Introductionmentioning

confidence: 99%

“…In the DPA, because the focus is on carcinogenicity, the recommended tests are the Ames Salmonella assay and the hepatocyte DNA repair assay of Williams (Table II). These assays both have reliable end points that are complementary (57) and metabolic parameters that provide broad bioactivation capability (62) and also are complementary. Other assays individually may be as effective as either of these (1) but add little to the combination of the 2 for detection of carcinogens.…”

Section: Introductionmentioning

confidence: 99%

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Safety Assessment of Pharmaceuticals: Examples of Inadequate Assessments and a Mechanistic Approach to Assuring Adequate Assessment

Williams

1997

Toxicol Pathol

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Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens

Cited by 251 publications

References 14 publications

Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice

Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice

Mutagenicity testing of diethylene glycol monobutyl ether.

Safety Assessment of Pharmaceuticals: Examples of Inadequate Assessments and a Mechanistic Approach to Assuring Adequate Assessment

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