Transcriptional activity of quinone methides derived from the tumor promoter butylated hydroxytoluene in HepG2 cells.

Butylated hydroxytoluene (BHT) is a pulmonary toxin and tumor promoter in mice presumably due to the formation of two quinone methides (QMs) that alkylate cellular nucleophiles. The activation of stress genes by these electrophilic metabolites was investigated with an assay system consisting of 14 recombinant cell lines derived from the human hepatoma line HepG2, each carrying a unique promoter or response element construct fused to the reporter gene for chloramphenicol acetyl transferase (CAT). The largest responses to QMs occurred in cells containing either the metallothionein IIA, glutathione S-transferase Ya, or 70 kDa heat shock protein promoter, or the xenobiotic response element. The other cell lines exhibited only small or no effects. These results are consistent with transcriptional activities reported for several other electrophiles known to undergo covalent interactions with proteins.

Acetaminophen metabolism and cytotoxicity in PC12 cells transfected with cytochrome P4502E1.

Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen bio-transformation by CYP2E1 stimulates this pathway.

Acetaldehyde-induced growth inhibition in cultured rat astroglial cells.

Due to the important role of glial cells in brain maturation and reports on delayed astroglial proliferation following ethanol exposition, it was of great interest to examine the effects of the primary metabolite of ethanol--acetaldehyde--on astroglial cell growth. This was carried out by examining biochemical parameters of astroglial cells cocultured with Chinese hamster ovary cell line (CHO) transfected with alcohol dehydrogenase (ADH), able to generate acetaldehyde from ethanol. Acetaldehyde generated from ethanol by ADH-transfected CHO cells had an inhibitory effect on the growth of astroglial cells as assessed by measuring marker enzyme activities and culture protein levels. Moreover, both acetaldehyde and ethanol altered cell cycle and increased astroglial superoxide dismutase activity. Additionally, acetaldehyde, but not ethanol, increased malondialdehyde levels in cultured astroglia. These results clearly show that acetaldehyde may participate in the development of the Fetal Alcohol Syndrome.

Mechanisms of acetaldehyde-mediated growth inhibition: delayed cell cycle progression and induction of apoptosis.

Chronic ethanol exposure has been associated with pleiotropic effects on cellular function in vivo and in vitro, including inhibition of growth. To date, it has been difficult to dissociate the primary effects of ethanol from the effects of ethanol metabolism, generation of acetaldehyde, and reducing equivalents. We have previously described the development of a Chinese hamster ovary cell line, A-10, which expresses a transfected murine-liver alcohol dehydrogenase. Cultures of these cells accumulate acetaldehyde due to the low level of aldehyde dehydrogenase. One noticeable effect of chronic acetaldehyde exposure, but not ethanol exposure, is the inhibition of cell growth. This study focuses on the mechanisms that underlie this growth inhibition. Our studies with the A-10 cell on the rates of [3H]thymidine incorporation and flow cytometry of asynchronous cultures indicated that acetaldehyde did not lead to arrest of the cell cycle in the G1 phase as has been found in other models of ethanol exposure. Rather, we observed a generalized delay in cell cycle progression. However, the slower cell cycle did not account exclusively for the slower rates of cell accumulation. Chronic exposure to acetaldehyde also increased the rate of cell death. The increased rate of cell death was both cumulative and dose-dependent. The dead cells accumulated in the medium and were apoptotic. Apoptosis was confirmed using morphological criteria and quantitation of DNA fragmentation. These data lend additional support to the idea that chronic acetaldehyde exposure can affect the mechanisms that regulate cell division and the apoptotic program.

Acetaldehyde exposure causes growth inhibition in a Chinese hamster ovary cell line that expresses alcohol dehydrogenase.

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.

Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression.

The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol.min-1.mg microsomal protein-1 vs 68 +/- 48 nmol.min-1.mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol.min-1.mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.

Characterization of the transport of a synthetic bile salt, iodinated cholyl-glycyl-tyrosine, in isolated cultured rat hepatocytes.

The uptake of tri-hydroxy conjugated bile salts by hepatocytes is principally by a sodium-dependent carrier. We examined the uptake kinetics of the high-specific-activity, hydroxylated, conjugated bile salt 125I-labeled cholyl-glycyl-tyrosine, to determine whether this synthetic bile salt was transported by the sodium-dependent bile salt system. 125I-labeled cholyl-glycyl-tyrosine was synthesized, and its transport kinetics were studied in freshly cultured rat hepatocytes. Uptake into hepatocytes was time and temperature dependent and was decreased by the inhibitors diisothiocyanodisulfonic acid stilbene, probenecid and carbonyl cyanide chlorophenyl hydrazone, demonstrating carrier mediation and energy dependence. At concentrations of iodinated cholyl-glycyl-tyrosine less than 10 mumol/L, uptake was 27% +/- 5% sodium dependent, whereas at concentrations from 10 mumol/L to 40 mumol/L uptake was 52% +/- 4% sodium dependent. The apparent affinity for uptake of 125I-labeled cholyl-glycyl-tyrosine was 8 +/- 2 mumol/L, and the maximal velocity was 50 +/- 20 pmol/micrograms DNA/min. Both taurocholate and indocyanine green inhibited uptake of 125I-labeled cholyl-glycyl-tyrosine. Indocyanine green inhibited the uptake of 125I-labeled cholyl-glycyl-tyrosine (Ki = 10 microns) more effectively than taurocholate (Ki = 20 microns). We conclude that 125I-labeled cholyl-glycyl-tyrosine is not a specific probe for either sodium-dependent bile salt or sodium-independent organic anion carriers, but appears to use both systems in a concentration-dependent manner in cultured rat hepatocytes.

trans-activation of viral enhancers including long terminal repeat of the human immunodeficiency virus by the hepatitis B virus X protein.

Human hepatitis B virus contains an open reading frame designated X. We have investigated the trans-activating function of the hepatitis B virus X gene in regulating transcriptional control elements. In the HBV genome the major target for X trans-activation is the enhancer element. Further, the X protein stimulates several other viral promoters/enhancers including the long terminal repeats (LTR) of human retroviruses. One of the viral sequences studied in detail is the human immunodeficiency viral (HIV) LTR which is trans-activated by the X protein. Using mutational analysis of the HIV LTR, we show that the NF-kappa B sequences contained within the U3 region are involved in this stimulatory activity. Nuclear run-on analyses support the notion that X-mediated trans-activation occurs at the level of transcription.

Expression of the hepatitis B virus X gene in mammalian cells.

The hepatitis B virus (HBV) DNA contains an open reading frame (ORF) designated X, which has the capacity to encode a protein of 16,560 Da (subtype adw). Such a protein has not been identified in either HBV particles or infected human livers, and therefore its role in the viral life cycle remains unknown. We report here the expression of the HBV X ORF in cultured cells using recombinant vectors. A protein of 16 kDa was identified by means of an antiserum prepared against a synthetic peptide and with human antisera from hepatitis B patients as well as those with hepatocellular carcinoma. Cell fractionation and immunofluorescence studies suggest a probable association with cytoskeletal components. Our studies further located a promoter sequence upstream of the X ORF, which directs the transcription of a 0.7- to 0.8-kilobase X-specific RNA in transfected human hepatoma cells.

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

Transcriptional control elements of hepatitis B surface antigen gene.

A series of recombinant plasmid vectors containing hepatitis B virus (HBV) DNA sequences was constructed to study the biosynthesis of the hepatitis B virus surface antigen (HBsAg) RNA and to locate transcriptional control elements involved in the regulation of the S and pre-S DNA sequences. We examined the transcription of the HBsAg gene in permanent cell lines that were developed by transfecting with recombinant vectors containing HBV sequences and the neomycin gene followed by G418 selection. We further defined the promoter activities upstream of and within the pre-S sequences using the assayable chloramphenicol acetyltransferase gene. Results obtained from S1 nuclease digestion and primer extension suggest that HBsAg transcripts are initiated at multiple sites in the pre-S region and from a site upstream of the pre-S region. Chloramphenicol acetyltransferase assays indicate that DNA sequences within and upstream of the pre-S region contain promoter activities and that the "TATA" sequence-containing promoter and the internal promoter show similar levels of activities in CV-1 cells and several other cell lines tested.

Purification of a HeLa cell receptor protein for group B coxsackieviruses.

Coxsackievirus B3 (CB3) firmly attaches to HeLa cells, forming a specific complex between the virus and its receptor on the cell surface. We extracted this virus-receptor complex (VRC) with the detergents sodium deoxycholate and Triton X-100. The VRC was identified by its sedimentation coefficient (140S), which was less than that of virions (155S). Formation of the VRC from cell lysates and 3H-CB3 occurred with the same specificity as did attachment of virions to cells, in that its formation was blocked by unlabeled CB3 but not by poliovirus. The VRC was purified 30,000-fold by differential and sucrose gradient centrifugation. Iodination with Na125I revealed that the purified VRC consisted of the normal CB3 proteins and one additional protein (RP-a) with an approximate molecular weight of 49,500. RP-a was eluted from the VRC and was shown to rebind with CB3 and CB1 virions but not with poliovirus type 1. We propose that Rp-a is a protein in the plasma membrane receptor complex which is responsible for the specific recognition and binding of the group B coxsackieviruses.

Radiochemical determination of polyamines in poliovirus and human rhinovirus 14.

HeLa cells were made strictly dependent upon polyamine by growth in the presence of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase. Under these conditions, the specific activity of the cellular polyamine pools eventually equilibrated to that of exogenously supplied [14C]putrescine; however, the process was very slow, requiring half-equilibration times of about 16 h for spermidine and 28 for spermine. Thus, the distribution of radioactivity in individual polyamines became a valid measure of polyamine content only after a continuous 4-day incorporation period. When propagated in polyamine-labeled cells, two picornaviruses were found to incorporate substantially different amounts of polyamine: about 0.6% of the cell pool for human rhinovirus 14 but only 0.04% for poliovirus. This content of polyamine was sufficient to neutralize nearly 27% of the negative charge of the RNA in human rhinovirus 14 but only 1.6% in poliovirus.