Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.

Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus.

The enzyme of rat liver that can transform N -hydroxy- N -2-fluorenylacetamide into a reactive derivative capable of introducing fluorenylamine groups into nucleic acids has been shown to be a sulfhydryl-dependent enzyme with a molecular weight of approximately 28,000. A 30-fold purification of the enzyme from 105,000 × g supernatants of liver has been achieved by precipitation with ammonium sulfate and gel filtration on Sephadex G-100. Evidence that this mechanism of activation involves transfer of the N -acetyl group to the oxygen of the hydroxylamine came from experiments that showed that O -methylation of N -hydroxy- N -2-fluorenylacetamide prevented activation of the hydroxamic acid.

Distribution studies demonstrated considerable acyltransferase activity in kidney, stomach, small intestine, and colon; lung and spleen were less active; and blood, brain, and muscle were essentially without activity.

Tissue distribution studies and protein purification experiments utilizing ammonium sulfate precipitation and gel filtration techniques disclosed that the enzyme responsible for the activation of N -hydroxy- N -2-fluorenylacetamide was inseparable from the enzyme that transfers the acetyl group of N -hydroxy- N -2-fluorenylacetamide to 4-aminoazobenzene.

Acyltransferase-catalyzed activation of N -hydroxy- N -2-fluorenylpropionamide demonstrated that the activation of hydroxamic acids was not restricted to acetylated derivatives.

Hepatic acyltransferase-catalyzed formation of fluorenylamine-substituted nucleic acid was inhibited by both arylamines and arylacetamides. This inhibition, considered with previous reports of the inhibition of N -hydroxy- N -2-fluorenylacetamide hepatocarcinogenesis by acetanilide and p -hydroxyacetanilide, suggest that acyltransferase-catalyzed activation of arylhydroxamic acids may be involved in the formation of liver tumors.

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Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acyltransferase.

The publication of several studies reporting genotoxic effects of paracetamol, one of the world's most popular over-the-counter drugs, has raised the question of regulatory action. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. There are, however, published data giving clear evidence that paracetamol causes chromosomal damage in vitro in mammalian cells at high concentrations and indicating that similar effects occur in vivo at high dosages. Available data point to three possible mechanisms of paracetamol-induced genotoxicity: (1) inhibition of ribonucleotide reductase; (2) increase in cytosolic and intranuclear Ca2+ levels; (3) DNA damage caused by NAPQI after glutathione depletion. All mechanisms involve dose thresholds. Studies of the relationship between genotoxicity and toxic effects in the rat (induction of micronuclei in rat bone marrow including dose-response relationship, biotransformation of paracetamol at different dosages, concomitant toxicity and biochemical markers) have recently been completed. These studies, which employed doses ranging from the dose resulting in human therapeutic peak plasma levels to highly toxic doses, give convincing evidence that genotoxic effects of paracetamol appear only at dosages inducing pronounced liver and bone marrow toxicity and that the threshold level for genotoxicity is not reached at therapeutic dosage. Reliable studies on the ability of paracetamol to affect germ cell DNA are not available. However, based on the amount of drug likely to reach germ cells and the evidence of thresholds, paracetamol is not expected to cause heritable damage in man. Various old and poorly designed long-term studies of paracetamol in the mouse and rat have given equivocal results. A few of these studies showed increased incidence of liver and bladder tumours at hepatotoxic doses. National Toxicology Program (U.S.A.) feeding studies have shown that paracetamol is non-carcinogenic when given at non-hepatotoxic doses up to 300 mg/kg/d to the rat and up to 1000 mg/kg/d to the mouse. Taking into account the knowledge of the hepatotoxicity and metabolism of paracetamol and the existence of thresholds for its genotoxicity, the animal studies do not indicate a carcinogenic potential at non-hepatotoxic dose levels. Based on this updated assessment of the genotoxicity and carcinogenicity of paracetamol, it is concluded that there is no need for regulatory action.

Series: current issues in mutagenesis and carcinogenesis, No. 65. The genotoxicity and carcinogenicity of paracetamol: a regulatory (re)view.

The expression of three cellular oncogenes (c-myc, c-Ha-ras, and c-delta-raf), the epidermal growth factor receptor gene, and two endogenous retrovirus-like sequences [rat leukemia virus (RaLV) and 30S] was examined in control rat livers and in 16 liver tumors. The tumors were induced in Sprague-Dawley male and female rats by a single i.p. injection of diethylnitrosamine at 1 or 2 days after birth, followed by dietary exposure to phenobarbital beginning at weaning. Increased expression of c-myc was seen in most of the tumors, but there was no consistent increase or decrease in expression of c-Ha-ras or c-delta-raf. It is of interest that a number of the tumor samples showed a decrease in epidermal growth factor receptor RNA. In all of the tumors, including both hepatocellular adenomas and carcinomas, there was a marked increase in expression of the endogenous RaLV sequence, and over 90% of the tumors displayed increased expression of the 30S endogenous retroviral-like sequence. No or a very low level of expression of the RaLV and 30S sequences was found in the control livers. The extent of expression of the RaLV and 30S sequences in individual tumors did not correlate with the extent of expression of c-myc or c-Ha-ras. Although increased expression of certain endogenous retrovirus-related sequences appears to be a common finding during rat liver carcinogenesis, the significance of this finding remains to be determined.

https://cancerres.aacrjournals.org/content/canres/47/13/3421.full.pdf

Expression of retroviral sequences and oncogenes in rat liver tumors induced by diethylnitrosamine.

Cancer "causation" by infections--individual contributions and synergistic networks.

An investigation into the possible relationship between chemical carcinogen induction of rat mammary tumors and the expression of an endogenous retroviral genome was initiated. Mammary tumors were induced in female SD rats with 7,12-dimethylbenz[a]anthracene (DMBA). Tumors, identified histologically as mammary adenocarcinomas, were analyzed for RNA of a replication-defective endogenous retrovirus or RNA of a helper-independent endogenous type C virus. Expression of RNA of the replication-defective virus was detected in mammary tumors weighing 0.2--2.0 g. Larger tumors, for which histologic examination revealed proportionally more fibroblastic tissue than epithelial cells, did not contain comparable concentrations of this viral RNA. RNA homologous to a helper-independent rat type C retrovirus was not detected in tumors of any size. A cell line was established from a primary DMBA-induced mammary adenocarcinoma and appeared similar to the small mammary tumors with respect to endogenous type C viral RNA expression. We discuss possible implications of the expression of endogenous replication-defective viruses for use as markers for the effects of chemical carcinogens.

Expression of RNA of an Endogenous Replication-Defective Retrovirus in Rat Mammary Adenocarcinomas Induced by 7,12-Dimethylbenz[a]anthracene

Effect of Acetanilide and p-Hydroxyacetanilide on the Carcinogenicity of N-2-Fluorenylacetamide and N-Hydroxy-N-2-fluorenylacetamide in Mice, Hamsters, and Female Rats

Martin L. Wenk

Papers

Expression of RNA of an Endogenous Replication-Defective Retrovirus in Rat Mammary Adenocarcinomas Induced by 7,12-Dimethylbenz[a]anthracene

Effect of Acetanilide and p-Hydroxyacetanilide on the Carcinogenicity of N-2-Fluorenylacetamide and N-Hydroxy-N-2-fluorenylacetamide in Mice, Hamsters, and Female Rats