Mutational specificity of aflatoxin B1. Comparison of in vivo host-mediated assay with in vitro S9 metabolic activation.

An intrasanguineous host-mediated assay was used to determine the pattern of mutagenesis induced by the carcinogen aflatoxin B1 in the lacI gene of Escherichia coli recovered from rat liver. To investigate the influence of different types of metabolic activation, the mutation spectrum induced by AFB1 activated in vitro by a commercially prepared S9 microsomal fraction from Aroclor 1254-treated rats was also obtained. A total of 281 forward mutations affecting the N-terminal region of the lacI gene were characterized by DNA sequencing analysis. AFB1 induced similar type of mutations with similar site specificity when activated by the standard S9 fraction or by employing a rat host-mediated assay. These results indicate the ability of the in vitro S9 fraction to mimic the in vivo metabolism, suggesting that the same active metabolite, presumably AFB1 8,9-epoxide, is responsible for generating a similar pattern of DNA damage, as reflected in the similarity of mutational spectra. For both activation systems, most mutations (>90%) were base substitutions that occurred primarily at G:C pairs. Somewhat over one-half of G:C targeted substitutions were GC>TA transversions, other mutations being evenly divided between G:C>AT transitions and GC>CG transversions. The mutational specificity exhibited by activated AFB1 can be explained by incorporation of different bases opposite a single type of non-instructive lesion during error-prone DNA synthesis. To what extent the mutations are due to the main adduct (AFB1-N7-Gua), its imidazole-ring-opened derivative or an apurinic site remains unknown.

Comparison of co-cultivation of V79 cells with rat hepatocytes and rat H4IIE hepatoma cells for studying nitrosamine-induced hprt gene mutations.

The induction of mutations by nitrosamines in the hprt locus of V79 Chinese hamster cells was examined after metabolic activation in a co-cultivation system using either freshly isolated rat hepatocytes or H4IIE rat hepatoma cells and the results obtained were compared with systems which employ the rat liver microsomal fraction (S9-mix). This study was also designed as a first approach to investigating the induction of point mutations by tobacco-specific nitrosamines in mammalian cells in order to obtain information about the significance of these compounds in connection with the carcinogenicity of tobacco smoke. The mutagenicity of two tobacco-specific nitrosamines, 4-(methylnitroso)-1-(3-pyridol)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were investigated and compared to two extensively investigated nitrosamines, i.e. dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). DMN was activated to mutagenic species by primary hepatocytes at mumolar concentrations, i.e. 1/100 of the concentrations required for mutagenesis by DEN and NNK. NNN was not activated to mutagenic species by liver S9 or primary hepatocytes. The findings shown here on the mutagenicities of NNK and NNN with liver preparations are in agreement with their relative carcinogenic potencies. When the established liver cell line H4IIE was used for metabolic activation, DMN and was found to be mutagenic, whereas the results for NNN were borderline and for DEN and NNK were without effect. The fate of these compounds via different metabolic pathways is discussed in terms of systems for detection of mutagenic metabolites and type of mutation induced.

Specificity of antibody-dependent cellular cytotoxicity in sera from human immunodeficiency virus type 2-infected individuals.

ADCC activity in sera from HIV-2-infected individuals was monitored against HIV-1IIIB, SIVmac, and three different HIV-2 strains. The sera mediated ADCC against the HIV-2 strains in high frequencies and reacted equally well with SIVmac, whereas no cross-reactivity was seen against HIV-1IIIB. The degree of antigenic similarities between the virus strains was also evaluated in order to estimate the variability of ADCC target regions. The SIVmac strain and two of the HIV-2 strains were antigenically more similar to each other whereas another HIV-2 strain appeared more distantly related with regard to ADCC target regions. Although strain-specific ADCC was present in some HIV-2-positive sera, HIV-2 ADCC was more broadly reactive and appeared in higher frequencies against these specific strains than has been previously shown for HIV-1 ADCC in a group of four HIV-1 strains. The difference was, however, not significant. To further delineate target regions for ADCC the sera were tested against peptides representing different regions of the HIV-2 envelope protein. The V3 region and the C-terminal end of gp125 were thus suggested to be involved in ADCC. Target regions for HIV-2-specific ADCC may only partly overlap with HIV-2-neutralizing regions since the two activities were not always present in the same sera. Characterization of broadly reacting immune responses like HIV-2-specific ADCC and identification of their specific target epitopes is essential for the development of an efficient AIDS vaccine.