Coinhibition of viral interferon induction by benzo[a]pyrene in association with occupation-related particles.

Benzo[a]pyrene (B[a]P) in combination with coal, asbestos, or metal particles was studied for its inhibitory effects on interferon-alpha/beta induction by influenza virus in rhesus monkey kidney (LLC-MK2) cell monolayers. B[a]P per se had no adverse effect on the induction process. However, when cell cultures were pretreated with B[a]P that was bioactivated by rat liver S9 homogenate, from 52 to 65% inhibition of interferon induction occurred. Significantly greater (P less than 0.05) depression (coinhibition) of viral interferon induction (greater than 83%) resulted when bioactivated B[a]P was incorporated with coal particles representative of coal rank (anthracite, bituminous, lignite, peat). Coinhibition affected by bioactivated B[a]P was coal rank-independent but any interferon inhibitory activity affected by coal particles per se was coal rank-dependent. When metals (aluminum, aluminum oxide, ferric oxide, nickel, or chromium) or asbestos fibers (chrysotile, crocidolite, anthophyllite, or amosite) were individually mixed with bioactivated B[a]P, coinhibition of cellular interferon synthesis also resulted which was significantly greater (P less than 0.05) than that manifested by bioactivated B[a]P or particles per se. Coinhibition of interferon induction by silicates (Min-U-Sil, DQ-12, hypersthene, or wollastonite) and the bioactivated hydrocarbon was not in evidence although some silicates alone partially inhibited the induction process. Viral interferon induction was inhibited in a dose-response manner by B[a]P (+/- S9) in combination with selected particles. The adsorption of B[a]P to all types of particles was no more than 5.98 micrograms B[a]P/mg of particles and, moreover, less than 0.5% by weight. These findings provide further evidence that bioactivated B[a]P and occupation-related particles act together to coinhibit a biological defense mechanism, the interferon induction phase of the interferon system.

Interferon production in rat type II pneumocytes and alveolar macrophages.

The time course and magnitude of interferon production induced by influenza virus were determined in type II pneumocytes and alveolar macrophages isolated from rats (Sprague-Dawley). Although the peak of interferon production was approximately 20 h in both alveolar cell types, it was more than three- to fourfold higher in type II cells than in macrophages. Dose-response relationships were noted between the virus multiplicity of induction as well as population numbers of either alveolar cell type and interferon yields. The viral-induced cytokines produced by rat type II cells and alveolar macrophages exhibit physiochemical and biological properties characteristic of interferons and, with respect to type II pneumocytes, mark their heretofore unrecognized capability to produce interferon. The best cross-species antiviral protection afforded by these rat interferons to cells of different origin, expressed as percentage of homologous species (100%), was as follows: guinea pig 50, mouse 40%, and both human and monkey 0%. The heterologous antiviral activity by interferons from either rat alveolar macrophages or type II cells on alpha interferon-sensitive guinea pig cells suggests that these cytokines may be more appreciably related to the alpha-like interferon species. The growth of influenza and Sendai viruses was precluded in both rat alveolar macrophages and type II pneumocytes. The findings herein suggest that type II cells may be a major source of alveolar interferon for activating the antiviral state and modulating alveolar cell functions requisite for lung integrity.

Benzo[a]pyrene: kinetics of in vitro bioactivation in relation to inhibition of viral interferon induction.

The kinetics of benzo[a]pyrene (BaP) bioactivation by rat liver S9 fraction was characterized on the basis of inhibition of influenza virus induction of interferon-alpha/beta (IFN-alpha/beta) in mammalian LLC-MK2 cell cultures. Both viral IFN induction and production phases were sensitive to the adverse effects of bioactivated BaP. The integral role of S9 for BaP bioactivation and the resultant inhibition of viral IFN induction was substantiated by dose-response relationships, time-dependency of effects, and reversibility of adverse reactivity. When preceded by the analog, benzo[e]pyrene (BeP), the inhibitive action of bioactivated BaP on IFN induction was abrogated. That the ability of exogenous IFN to confer antiviral cellular resistance was unaffected by bioactivated BaP indicates that neither requisite cellular protein nor enzyme syntheses were impaired. In cells pretreated with bioactivated BaP, influenza virus multiplication reached a level that was more than twofold higher than in normal cells which was a reflection of decreased IFN production. These findings further imply that neither virus inducer-cell interactions (attachment and penetration) nor requisite viral protein and RNA syntheses were affected appreciably. BaP was selectively cyto-antagonistic to critical inducer-processing phases of IFN induction. Of 32 different mammalian cell cultures tested for indigenous metabolizing enzyme-bioactivation of BaP, based on approximately equal to 50% resultant inhibition of IFN induction, only 37.5% were responsive.

Interferon induction inhibition and mutagenic activity of nitrosated coal dust extract.

Specified cytotoxicity and mutagenicity of coal dust extract (mixture of solvent extractions of bituminous coal nitrosated by NaNO2) were investigated because of the association of an excess risk of gastric cancer in coal miners. The effect of nitrosated coal dust on a cellular defense component of the interferon system, the induction of interferon (alpha/beta) in mammalian cell cultures by influenza virus, and mutagenicity, using the Salmonella/microsome assay, were determined. Nitrosated coal dust extract contained both bioactivation-independent and -dependent (microsome enzymatic activation) compounds that significantly inhibited the viral induction of interferon by greater than 50%. Nitrosated extract was mutagenic but exhibited no increase in mutagenic activity in the presence of microsomal enzymes. With further extraction of nitrosated coal dust extract by horse serum and fractionation thereof, the soluble chemical complexes formed with fractions of high molecular weight without bioactivation were dominant in both mutagenicity and inhibition of interferon induction. Low-molecular-weight fractions, with or without a metal chelator, and with or without bioactivation, all inhibited interferon induction comparably and significantly. There was no mutagenic activity manifested by these serum fractions. Metal-serum complexes were either not formed, or, if present, were ineffectual according to the biologic criteria employed. The findings of this study are discussed in terms of the association between nitrosated coal dust and gastric carcinogenesis.

Silicate minerals and the interferon system.

Natural-occurring minerals representative of six silicate classes were examined for their influence on interferon induction by influenza virus in Rhesus monkey kidney (LLC-MK2) cell monolayers. Minerals within the classes nesosilicate, sorosilicate, cyclosilicate, and inosilicate exhibited either little or marked (50% or greater) inhibition of interferon induction. Within the inosilicate class, however, minerals of the pyroxenoid group (wollastonite, pectolite, and rhodonite) all significantly showed a two- to threefold increase in interferon production. Silicate materials in the phyllosilicate and tectosilicate classes all showed inhibitory activity for the induction process. When silicate minerals were coated with the polymer poly(4-vinylpyridine-N-oxide), the inhibitory activity of silicates on viral interferon induction was counteracted. Of nine randomly selected silicate minerals, which inhibited viral interferon induction, none adversely affected the ability of exogenous interferon to confer antiviral cellular resistance. Increased levels of influenza virus multiplication concomitant with decreased levels of interferon occurred in cell monolayers pretreated with silicates. The findings of this study demonstrate the diverse effects of minerals representative of different silicate classes on the interferon system and indicate that certain silicates in compromising the viral interferon induction process may increase susceptibility to viral infection.

Benzo[a]pyrene metabolites: effects on viral interferon induction.

Benzo[a]pyrene (BaP) metabolites were assessed, with and without enzymatic activation by rat liver S9, for their inhibitory activities on influenza virus induction of interferon-alpha/beta (IFN-alpha/beta) in mammalian (LLC-MK2) cell cultures. Although BaP per se was inactive, metabolized BaP reduced viral IFN induction by approximately 80%. BaP metabolites (phenols, diols, 6-substituted derivatives) exhibited significant inhibitory activity (50% or greater) only when they were activated enzymatically. Although not significant, the diol metabolites without activation mildly reduced IFN induction on the average of 32%. The quinones did not adversely affect the IFN induction process, but three of the seven metabolites tested showed approximately 30% inhibitory activity in the presence of S9. BaP diol epoxides were direct inhibitors of viral IFN induction while derivatives of these epoxides, tetrols and triols, showed negligible inhibition even with S9. In general, the reported microbial mutagenicities of BaP metabolites could be correlated with their abilities to inhibit IFN induction. That activation-dependent hydrocarbons can be metabolized by S9 added to mammalian cell cultures resulting in the inhibition of viral IFN induction extends the capability and credibility for assessing suspect mutacarcinogens on this basis.

Coinhibition of viral interferon induction by benzo[a]pyrene and chrysotile asbestos.

Benzo[a]pyrene (B[a]P) and its noncarcinogenic analog, benzo[e]pyrene (B[e]P), each in combination with chrysotile, were studied for their inhibitory effects on interferon (IFN) induction by influenza virus in rhesus monkey kidney (LLC-MK2) cell monolayers. B[a]P alone had no adverse effect on IFN induction; however, from 60 to 70% inhibition of IFN production occurred when B[a]P was enzymatically activated by rat liver S9. Chrysotile's inhibitory effect on the IFN process was similar in magnitude to that of activated B[a]P. The combination of activated B[a]P with chrysotile resulted in coinhibition of IFN induction which significantly exceeded (P less than 0.05) the inhibitory activity of the reagents tested alone or in other combinations. B[e]P alone or with S9 neither affected IFN induction nor was it capable of further enhancing chrysotile's inhibition of IFN synthesis. These findings provide further evidence of enhanced deleterious action by the combination of asbestos and activated B[a]P on a biological defense mechanism and further support the discriminatory power and credibility of the inhibition IFN induction assay for evaluating potential carcinogens.

In vitro biologic responses to native and surface-modified asbestos.

A comparative study was made of in vitro biologic responses to native chrysotile, amosite, and crocidolite and corresponding asbestos fibers whose surfaces were modified by metal oxides. Interferon induction by influenza virus was depressed by approximately 50% by all native asbestos whereas corresponding surface modified asbestos minimally affected this nonspecific cellular defense mechanism. The release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), and lysosomal enzymes, beta-N-acetylglucosaminidase (beta-NAG) and beta-glucuronidase (beta-Gluc), by rat alveolar macrophages after exposure to either native or surface-modified asbestos (which is indicative of membrane damage) was monitored. Although both native and surface-modified asbestos induced significant leakage of LDH, generally, lesser amounts of the enzyme were released as a result of exposure to the latter than to native asbestos. Whereas all forms of native asbestos caused significant release of beta-NAG and beta-Gluc, leakage of these enzymes from macrophages exposed to surface-modified asbestos was minimal. In contrast to native asbestos which induced irritation of cell membranes, as indicated by hemolysis of sheep erythrocytes, surface-modified asbestos exhibited minimal hemolytic activity. The findings indicate that surface modification of different asbestos by metal oxides generally lessened the adverse effect of the native mineral on the aforementioned biologic entities.

Inhibition of viral interferon induction in mammalian cell cultures by azo dyes and derivatives activated with rat liver S9 fraction.

Four azo dyes (Benzopurpurine 4B, trypan blue, Direct Blue 15, and Congo red) and their derivatives (o-tolidine, o-dianisidine, and benzidine) were studied for their effect on induction of interferon by influenza virus in mammalian, Rhesus monkey kidney (LLC-MK2), cell monolayers. Whereas Benzopurpurine 4B, Direct Blue 15, and Congo red inhibited viral interferon induction from approximately 35 to 60%, negligible inhibition was noted with trypan blue and other derivative compounds. By comparison, when rat S9 fraction was used for enzymatic activation of azo dyes and derivatives, interferon inhibition was significantly depressed moreover (P less than 0.01 to less than 0.0001) by all chemical compounds. The concentration of rat S9 (0.5%) used for metabolic activation of dyes and congeners was critical because concentrations greater than 0.5% of S9 per se inhibited the interferon induction process. Uninduced hamster S9 and both Aroclor 1254-induced hamster and rat S9 fractions were all comparable in their ability to activate the chemical compounds tested. That potential mutagenic and carcinogenic chemicals which require metabolic activation can be discriminated on the basis of interferon induction inhibition in eukaryotic cell cultures augurs for the usefulness and credibility of this system.

Influenza virus infection in mice after exposure to coal dust and diesel engine emissions.

Influenza virus infection initiated after aerosol exposure of CD-1, white Swiss mice for durations of 1, 3, and 6 months to respirable particulates maintained at 2 mg/m3 of either coal dust (CD), diesel engine emissions (DEE), a combination of both (CD/DEE), or to filtered air (control) was studied. The course of infection in mice previously exposed for 1 month to various particulates did not differ appreciably among the four animal groups with respect to mortality, virus growth in lungs, interferon levels, or hemagglutinin antibody response. In mice exposed for 3 and 6 months to different particulates, the mortality response was similar among all animal groups. However, the percentage of animals showing lung consolidation was significantly higher in the 3-month groups exposed to DEE (96.5%) and CD/DEE (97%) than in the control (61.2%); in the 6-month groups, the percentages were twice that of the control for both DEE- and CD/DEE-exposed animals. Complementing these observations of both 3- and 6-month-exposed animals was the higher virus growth levels attained in the DEE and CD/DEE animals with concomitant depressed interferon levels which were the inverse of findings noted in the control group. Hemagglutinin-antibody levels in particulate-exposed animals, especially at the 6-month interval, were fourfold less than the control. Histopathologic examination of lungs revealed no qualitative differences in the inflammatory response at any one specified time interval of exposure to influenza virus among the control and particulate-exposed animal groups. However, there were differences in severity of reaction in relation to the particulate component of the exposures. Focal macular collections of pigment-laden macrophages were seen only in DEE and CD/DEE but not in CD animals after 3- and 6-month exposures. The findings of this study indicated that the severity of influenza virus infection is more pronounced in mice exposed to diesel engine emissions than in control animals and it is not appreciably accentuated by coal dust.

Effect of chromium and manganese particles on the interferon system.

Mammalian (LLC-MK2) cell monolayers pretreated with either chromium or manganese particles depressed viral induction of IFN by approximately 50% but the presence of metal particles did not prevent exogenous IFN from conferring antiviral cellular resistance. Manganese particles were more detrimental to viral IFN induction than chromium particles in that almost tenfold less of the former achieved a comparable magnitude of IFN inhibition. Although rates of influenza virus multiplication in either chromium or manganese-treated and control cell cultures were similar, virus attained a level of growth almost twofold higher in metal-treated cells than in controls. This was related to suppression of viral IFN induction by metal particles. Neuraminidase treatment of cell surface salioglycoproteins or pretreatment of chromium or manganese particles with sialic acid abrogated the adverse activity of metal particles on viral IFN induction. These findings suggest that the receptivity and interaction of cell membrane-bound sialic acid residues with metal particles are involved in the altered cellular protective response described.

Effects of lignite fly ash particulates and soluble components on the interferon system.

Induction of interferon by influenza virus was depressed by approximately 50% when mammalian (LLC-MK2) cell monolayers were pretreated with lignite fly ash. The presence of fly ash, however, did not impair the ability of exogenous interferon to confer antiviral cellular resistance. Influenza virus multiplication in cell monolayers pretreated with fly ash attained a twofold higher level of growth than that noted in normal cell monolayers. This was related to suppression of viral interferon induction by fly ash. Whereas aqueous extracts of fly ash had no adverse effect on interferon induction, extractions of fly ash by either polar or nonpolar solvents, by horse serum with or without EDTA (a metal chelator), and fractionation of serum extracts yielded corresponding compounds, most likely organic and inorganic, that were antagonistic to viral interferon induction. Residual fly ash particulates after extraction by horse serum with EDTA were still capable of inhibiting viral induction of interferon. These findings indicate that several soluble components inherent to lignite fly ash and the particulate matrix per se may modify, independently or in concert, cellular defense behavior. Neither polar, nonpolar, nor horse serum extracts of lignite fly ash, however, showed mutagenic activity as determined by the Salmonella histidine reversion assay. Removal of cell-membrane-bound sialic acid (N-acetylneuraminic acid) by neuraminidase or pretreatment of lignite fly ash with sialic acid abolished the adverse activity of fly ash on viral interferon induction. This suggests that the interaction of cell-membrane-bound sialic acid residue with fly ash particulates may be involved in the altered state of cellular behavior described in response to viral induction of interferon.

Effect of coal rank on the interferon system.

Studies on the induction of interferon by influenza virus in monkey kidney (LLC-MK2) cell monolayers pretreated with coal dust revealed that the inhibitory activity of the mineral on this process was related to coal rank. Maximal inhibition of viral interferon induction was noted with high rank coal and the degression of this activity was related to coal's position in the carboniferous series; anthracite, bituminous, subbituminous, lignite, and peat. The cytocidal activity of each rank of coal, however, was comparable in relation to the corresponding quantities of coal dust that were tested indicating that findings related to interferon production are not explicable on the basis of remaining viable cells. Coal dust, irrespective of rank, also did not affect the ability of exogenous interferon to confer antiviral cellular protection. An inverse relationship mediated by coal of different rank occurred between influenza virus growth and interferon levels which suggested that increased virus growth reflected the ability of higher rank coal to affect adversely interferon production.

Action of antischistosomal drugs, hycanthone and its analog 1A-4 N-oxide, on viral interferon induction.

Two antischistosomal drugs, hycanthone and its indazole analog 1A-4 N-soxide, of which the former is a potent mutagen, differed markedly in their ability to depress interferon induction by influenza virus in rhesus monkey kidney (LLC-MK2) cell cultures. At the concentration of 10 micron/ml, hycanthone reduced interferon yields as much as 73%; the same quantity of analog had no adverse effect on interferon induction. Pretreatment of cell cultures with the analog negated the inhibitory activity of hycanthone on viral interferon induction; however, this did not occur when the sequence was reversed. Interferon-mediated antiviral cellular resistance was not affected when cell cultures were pretreated with either hycanthone or 1A-4 N-oxide. A possible association may exist between the mutagenicity of antischistosomal drugs and their ability to affect interferon synthesis.

Enhanced viral interferon induction by the mineral wollastonite.

The in vitro activity of the fibrous mineral wollastonite (CaSiO3) on the interferon system was investigated. Wollastonite enhanced the induction of interferon by influenza virus in mammalian (LLC-MK2) cell monolayers but the mineral per se did not induce interferon. The magnitude of enhanced interferon induction was dependent on mineral concentration, particle size, and its time and sequence of addition onto cell monolayers. A "synergistic effect" on viral induction of interferon was noted when cell cultures were interferon-primed and then treated with wollastonite. Interferon yields were significantly higher than those obtained by the use of either the primer or wollastonite alone. That influenza virus multiplied in wollastonite-treated cells to a level that was sevenfold less than that in normal cells was associated with increased interferon production. The ability of interferon to confer antiviral cellular resistance was not impaired by wollastonite. The findings of this study suggest that the incorporation of wollastonite in appropriate interferon inducer-host cell systems may be useful for augmenting interferon production.

Aflatoxin inhibition of viral interferon induction.

The inhibitory effect of four basic aflatoxins on interferon induction by influenza virus in LLC-MK2 cell monolayers follows a structure-activity series with decreasing potency in the order aflatoxin B1 greater than G1 greater than B2 approximately G2. Of the four aflatoxins, B1 was the most deleterious to both cell growth and the viability of cells in confluent cultures. The fact that higher levels of influenza virus growth were attained in aflatoxin-treated cells than in normal cell monolayers was related to increased aflatoxin concentration in association with decreased interferon production. The ability of interferon to confer cellular resistance against viral infection, however, was not altered by aflatoxin. The inhibitory activity of aflatoxin on interferon production may be a factor contributing to impairment of host resistance to viral infections.