Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.

Autocrine activity of interleukin 6 secreted by hepatocarcinoma cell lines.

Among several rat hepatoma cell lines known to secrete interleukin 6 (IL6), the HTC.JZ1 line stands out as a high-level producer. HTC.JZ1 cells were stimulated to secrete up to fourfold increased amounts of IL6 over 24 hours by treatment with lipopolysaccharides (LPS). Both functional IL6 levels, measured as hepatocyte stimulating factor (HSF) activity, and IL6 mRNA concentrations were increased proportionally by exposure to LPS. Similarly, IL6 mRNA was induced by LPS treatment in cultured primary rat hepatocytes. The induction of Il6 mRNA by LPS was inhibited both in primary hepatocyte and hepatoma cell cultures by treatment with the synthetic glucocorticoid dexamethasone, consistent with the known analogous repression of the IL6 gene by dexamethasone in macrophages, monocytes and fibroblasts. IL6 secreted by HTC.JZ1 cells was utilized as an autocrine inducer of endogenous acute phase gene expression: HTC cells expressed constitutive levels of alpha 2-macroglobulin (alpha 2M) mRNA specified by the major rat acute phase gene, the alpha 2M gene, which is known to be regulated by IL6. By contrast, normal rat liver biopsy material and a number of other rat hepatoma cell lines lacked endogenous IL6 production and showed very low to zero expression of endogenous alpha 2M mRNA. Expression of alpha 2M mRNA in HTC.JZ1 cells was inducible by treatment with LPS. The constitutive and the LPS-induced production of alpha 2M mRNA were significantly reduced (up to 50% inhibition) by addition of an anti IL6 serum to the culture medium and removal of the immune complexes. However, complete neutralization of the alpha 2M-inducing HSF activity could not be obtained with anti-IL6 serum alone, probably because HTC.JZ1 cells secrete comparable quantities of a second HSF activity. This activity, the cytokine leukemia inhibitory factor (LIF), is also known to stimulate transcription of the rat alpha 2M gene but was not reactive with anti-IL6 sera. The induction of IL6 mRNA in HTC cells by LPS was regulated at the transcriptional level, as demonstrated by a series of mutagenesis and transfection experiments. Progressive deletion of 5' flanking sequences from the IL6 gene promoter region reduced the basal level, and the LPS-induced promoter activity after transfection into HTC.JZ1 hepatoma cells. IL6 has been shown to act as an autocrine regulator of growth for certain B lymphoid cell lines derived from human multiple myelomas. The results presented here establish that IL6 secreted by certain hepatoma cell lines also acts in an autocrine fashion to induce expression of the endogenous acute phase alpha 2M gene.

Interleukin 6 is a negative regulator of the acute phase alpha 1-inhibitor III gene.

The broad-range proteinase inhibitor alpha 1-inhibitor III (alpha 1I3), a member of the complement C3/alpha 2-macroglobulin protein family, is the prototype of a negatively regulated acute phase protein. During an acute inflammatory reaction alpha 1I3 plasma protein and liver mRNA concentrations are decreased three- to fourfold in rats, and in chronic inflammations the protein concentration is reduced between ten- and 20-fold. In search of a cell culture model to study the regulation of the alpha 1I3 gene by mediators of inflammation, five well-established rat hepatoma cell lines were examined. All five lines constitutively expressed the gene, a marker for a highly differentiated hepatic phenotype, although at less than one-tenth the level of its expression in vivo. In the three hepatoma lines FAZA, FTO2B and FAO1, alpha 1I3 mRNA was decreased by treatment with interleukin 6 (IL6) and glucocorticoids. Among these lines untreated FAO1 cells produced the highest constitutive concentrations of alpha 1I3 mRNA and in FAO1 cells alpha 1I3 mRNA concentrations were decreased up to fourfold in a dose-responsive and time-dependent manner after treatment with IL6 alone or with combinations of IL6 and the synthetic glucocorticoid dexamethasone. Thus, IL6 alone was sufficient to negatively regulate alpha 1I3 mRNA levels in hepatoma cells with similar characteristics as occur during an inflammatory response in the liver. A number of other acute phase mRNA species, including alpha 1-acid glycoprotein, T2-kininogen, gamma-fibrinogen and alpha 2-macroglobulin were induced to higher levels by the same hormonal treatments in FAO1 cells. The fourfold reduction of alpha 1I3 mRNA concentrations in FAO1 cells could be reversed by treatment with 1 microM of a water-soluble derivative of forskolin, an activator of the cyclic AMP pathway. Thus, the effect of IL6 on the expression of the alpha 1I3 gene may involve the activation of the cyclic AMP pathway. In contrast, T2 kininogen mRNA levels were not altered by treatment of FAO1 cells with forskolin, suggesting that IL6 may act on this gene through a different mechanism.

Production of interleukin 6 by hepatoma cells.

Five rat hepatoma cell lines were shown to secrete hepatocyte-stimulating factors (HSFs) capable of inducing a characteristic spectrum of acute phase genes. Three of these lines, but not normal rat livers or livers from rats with an experimentally induced acute inflammation, produced interleukin 6 (IL6) mRNA. An anti-rat IL6 serum was prepared against synthetic peptides derived from the rat IL6 cDNA sequence. This antiserum cleared authentic rat IL6 and a fraction of the HSF activities secreted by the hepatoma cell lines. After concentration of culture supernatants from FTO2B hepatoma cells, IL6 was detected with the anti-IL6 serum by immuno-blot analysis. Biosynthesis of IL6 in the HTC line was demonstrated by metabolic labeling and immunoprecipitation. Secretion of HSF activities by hepatoma cells was increased by serum factors. These data suggest that different rat hepatoma lines each secrete different characteristic sets of HSF activities and establish unambiguously that IL6 is secreted by at least some of these lines.

Absorption of nicotine from a cigarette that does not burn tobacco.

Ten smokers participated in a study to compare the absorption of nicotine from the smoke aerosol of a new cigarette that heats, but does not burn tobacco (test) with a cigarette that burns tobacco (reference). The average plasma nicotine concentrations obtained by the 7th test cigarette (13 ng/ml) and 7th reference cigarette (24 ng/ml) were proportional to the nicotine yielded by the two cigarettes as determined under Federal Trade Commission machine-smoking conditions. These data demonstrate that the smoke aerosol obtained by smoking a cigarette which heats tobacco produces plasma profiles of nicotine that are similar to the profiles obtained from smoking a cigarette that burns tobacco.