Brian A. Laishes

Toxicity of aflatoxin B1 in rat and mouse hepatocytes in vivo and in vitro

The reported LD50 for the adult, male Fisher rat is 1.2 mg aflatoxin B1/kg body weight (i.p.); we have found that male C57BL/6 mice survive single doses of aflatoxin B1 as high as 60 mg/kg (i.p.). We have demonstrated a 1000-fold greater LC50 of aflatoxin B1 for primary mouse liver cell cultures from C57BL/6 male mice (3 X 10(-5) M) than for primary liver cells from F344 male rats (3 X 10(-8) M). In 4 h of exposure to a non-toxic dose (1 X 10(-9) M) of [3H]aflatoxin B1, cultured rat liver cells accumulated up to 5-fold higher concentrations of 3H label than did mouse liver cells. The difference in cell-associated counts was due largely to higher levels of aflatoxin B1 metabolites bound to macromolecules in the rat cells.

A LIVER COLONY ASSAY FOR A NEW HEPATOCYTE PHENOTYPE AS A STEP TOWARDS PURIFYING NEW CELLULAR PHENOTYPES THAT ARISE DURING HEPATOC ARCINOGENESIS

The architecture of premalignant epithelial tissues, in both the human and experimental animals, is often severely distorted by neoplastic nodules8 of tissue long before the appearance of frank malignancy.’ The proposal that cells contained within neoplastic nodules may in fact be the progenitors of malignant cells is a key hypothesis of cancer biologists.’ Thus, an understanding of the biological and biochemical properties of cells that form neoplastic nodules in epithelial tissues may reveal the mechanism by which only selected, focal areas of a tissue become malignant. Our approach to the study of neoplastic nodules is to utilize the rat liver model of chemically induced carcinogenesis,’ which is amenable to extensive technical manipulations. First, presumptive premalignant lesions can be readily controlled to appear in specified numbers with minimal asynchrony in the right lateral and caudate lobes of the liver.3 Secondly, during the premalignant stage of hepatocarcinogenesis, the liver tissue can be readily dissociated into its component cells:b some of which carry high concentrations of a “marker” enzyme, y-glutamyl transpeptidase (y-GT).‘ Thus, hepatocytes can be isolated free from one another to permit the identification of individual hepatocytes expressing new phenotypic properties that may be critical to the development of hepatocellular carcinoma. Thirdly, viable putative premalignant hepatocytes can be transferred surgically, via the hepatic portal circulatory system, to the livers of syngeneic host rats where macroscopic, y-GT-positive hepatocyte colonies can be generated within 10 days of the cell transfer pera at ion.^ The efforts of our laboratory are focused on identifying the cellular components that are essential to the carcinogenic process in the livers of rats treated with chemical hepatocarcinogens. Through the development of methods for obtaining critical target

DNA adduct formation and removal in N-acetoxy-2-acetylaminofluorene-exposed cultured cells and in organs from rats fed 2-acetylaminofluorene.

The development of carcinogen DNA-adduct antibodies has made possible a new approach to investigate carcinogen-DNA interactions (1). To quantitate adducts of a particular carcinogen, highly-avid rabbit antibodies have been employed to allow detection by radioimmunoassay (RIA) of one adduct in 10 5DNA bases (1). The studies described here employed the antiserum anti-guanosin-(8-yl)-acetylaminofluorene (anti-G-8-AAF) elicited against the nucleoside-adduct coupled covalently to bovine serum albumin and injected into rabbits (2). The antiserum is specific for the acetylated and deacetylated C-8 adducts of 2-acetylaminofluorene (2-AAF) with DNA (dG-8-AAF and dG-8-AF) (see Figure 1). It does not cross-react with the minor adduct, 3-deoxyguanison(N2-yl)-acetylaminofluorene (dG-N2-AAF) (see Figure 1), the carcinogen alone, or DNA (2,3). Since the C-8 adducts comprise the major proportion (80 to 90%) of adducts formed upon interaction of 2-AAF, or its activated derivative N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) with DNA in vivo (3,4,5), the anti-G-8-AAF was considered appropriate for initial studies. The antibody has been utilized to distinguish between the acetylated and deacetylated C-8 adducts of 2-AM in DNA, and to quantitate the proportions of each in DNA extracted from either cultured cells exposed to N-Ac-AAF or from livers and kidneys of male rats fed 2-AAF.

ELECTROPHORETIC RESOLUTION OF TWO RAT CLASS I ALLOANTIGENS EXPRESSED ON (WF x F344)F1 LIVER CELLS IN PRIMARY CULTURE

Polyvalent alloantisera, prepared by reciprocal immunization of F344 (RT1lv1 haplotypes) and WF (RT1u haplotype) rats, as well as monoclonal antibodies, were used to immunoprecipitate class I alloantigens from detergent extracts of monolayer cultures of 35S‐methionine‐labelled liver cells. Two‐dimensional IEF/SDS‐PAGE gel analysis resolved the RT1.Alv1 and RT1.Au class I antigens expressed on the liver cells in culture.

Rat Alloantigens as Cellular Markers for Hepatocytes in Genotypic Mosaic Livers During Chemically Induced Hepatocarcinogenesis

Genotypic mosaic rat livers were constructed by intravenous transplantation of carcinogen-altered F344 donor liver cells into livers of (WF × F344)F1 host rats. Donor rats were treated with a carcinogenic regimen consisting of diethylnitrosamine (200 mg/kg i.p.), followed by an experimental regimen of dietary 0.02% 2-acetylaminofluorene (AAF) and two-thirds partial hepatectomy (PH) (AAF/PH regimen). Host rats received the AAF/PH regimen in addition to transplanted donor liver cells. Utilizing alloantiserum specific for the WF major histocompatibility complex haplotype, RT1u, 97% of the γ-glutamyltranspeptidase-positive (GT+) liver colonies detected in cryostat sections of host rat livers 10–13 days after transplantation were shown to be of F344 donor origin (Hunt et al., Cancer Research, 42:227–236, 1982). Hepatocytes isolated from such genotypic mosaic livers were stained in suspension histochemically and with alloantisera by indirect immunofluorescence to localize GT+ phenotype and fluorescence in individual hepatocytes: 97% of GT+ hepatocytes were of F344 origin, consistent with the cryostat section results. Hepatocellular carcinomas arising in genotypic mosaic host rat livers 17 months after donor liver cell transplantation are presently being typed with alloantisera to establish the donor or host origin of the tumor cells.