Biological effect monitoring in industrial workers from the Czech Republic exposed to low levels of butadiene

Blood samples were collected twice (in 1993 and 1994) from 19 workers exposed to 1,3-butadiene and 19 matched controls. Three exposed and three control subjects were the same in 1993 and 1994. Personal passive dosimetry was performed in 1993 and twice in 1994 on the day preceding blood sampling. Mean exposure level in 1994 was 1.76 +/- 4.20 ppm (S.D.) and individual exposure levels ranged between 0.012 ppm (detection limit) and 19.77 ppm. Using the clonal assay, geometric mean of hprt mutant frequencies adjusted for cloning efficiency, age and smoking were, respectively, 7.85 (+/- 7.09) x 10(-6) and 10.14 (+/- 9.16) x 10(-6) in pooled (1993 plus 1994) exposed and control subjects. The difference was not statistically significant indicating that 1,3-butadiene did not induce a detectable increase in mutations at the hprt locus. A similar result was obtained for the 1994 subjects alone. There was no difference between adjusted geometric mean mutant frequencies of exposed and unexposed non-smokers or between exposed and unexposed smokers. Analysis of chromosomal aberrations in lymphocytes from 1994 subjects indicated that the percentage of aberrant cells was significantly enhanced in exposed subjects. In 1993 (data not shown), it was impossible to demonstrate a significant increase of aberrant cells in subjects exposed to 1,3-butadiene. Frequencies of micronuclei in cytochalasin-B blocked binucleate lymphocytes in exposed and unexposed 1994 subjects were not significantly different. This was also the case for earlier samples analyzed in the same plant. Using the comet assay for 1994 subjects, no statistically significant difference was found between the whole group of exposed and unexposed subjects. This was true for both the comet tail length and the percentage of DNA in the tail. In exposed smokers, however, the comet tail length was significantly longer than in unexposed smokers. Unexpectedly, in unexposed smokers the tail length was significantly shorter than in unexposed non-smokers. It was also unexpected that the percentage of DNA in the comet tail was significantly lower in exposed non-smokers than in unexposed non-smokers.     

Biochemistry of 1,3-butadiene metabolism and its relevance to 1,3-butadiene-induced carcinogenicity

Recently, the roles of specific P450 isoforms, myeloperoxidase (MPO), GSH-S-transferase and epoxide hydrolase in the metabolism of 1,3-butadiene, and its major oxidative metabolite, butadiene monoxide (BM), were investigated. The results provided evidence for P450s 2A6 and 2E1 being major catalysts of 1,3-butadiene oxidation in human liver microsomes. cDNA-expressed human P450s 2E1, 2A6, and 2C9 catalyzed BM oxidation to meso- and (+/-)-diepoxybutane (DEB), but the rates of BM oxidation in mouse, rat, or human liver microsomes were much lower than the rates of 1,3-butadiene oxidation in these tissues. Human MPO catalyzed 1,3-butadiene oxidation to BM, but MPO incubations with BM did not yield DEB. Rates of BM formation in mouse and human liver microsomes were similar and were nearly 3.4-fold higher than that obtained with rat liver microsomes. However, rat liver epoxide hydrolase activity was nearly 2-fold higher than that of mouse liver microsomes. Rat and mouse liver GSH-S-transferases exhibited similar BM conjugation kinetics, but rats excreted more BM-mercapturic acids compared to mice given low equimolar doses of BM. BM reacted with guanosine and adenosine to yield N7-, N2-, and N1-guanosinyl and N6-adenosinyl adducts, respectively. These results may contribute to a better understanding of the biochemical basis of 1,3-butadiene-induced carcinogenicity.     

Critical care management of the heart failure patient in the home

As part of a program to determine the underlying factors responsible for genotoxicity and perhaps lung cancer risk in Chinese women, we qualitatively identified the volatile components emitted during the heating of cooking oils to 265 degrees C. 1,3-Butadiene, benzene, and a series of aldehydes, olefins, and saturated hydrocarbons were elucidated in vapors from Chinese rapeseed oil. On a relative basis, the intensity of 1,3-butadiene vapors from this were 15.7-, 6.3-, and 1.4-fold greater than in the vapors from peanut, soybean, and Canola oils, respectively. Thus, the Chinese rapeseed oil yielded a higher emission rate of 1,3-butadiene than the other three oils investigated. The benzene formation rate followed a similar trend, i.e., its intensity in Chinese rapeseed oil was 14-, 6.6-, and 1.7-fold greater than in vapors from peanut, soybean, and Canola oils, respectively.     

Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1, 3-butadiene-exposed workers

The association of occupational exposure to 1,3-butadiene (BD) and induction of cytogenetic damage in peripheral lymphocytes was studied in 19 male workers from a monomer production unit and 19 control subjects from a heat production unit. The exposure to BD was measured by passive personal monitors. The following biomarkers were used: chromosomal aberrations (CA), sister chromatid exchanges (SCE), cells with a high frequency of SCE (HFC), micronuclei, comet assay parameters like tail length (TL) and percentage of DNA in tail [T (%)] and polymorphisms of GSTM1 and GSTT1 genotypes. BD exposure with a median value of 0.53 mg/m3 (range: 0.024-23.0) significantly increased (a) the percentage of cells with chromosomal aberrations in exposed vs. control groups (3.11% vs. 2.03%, P<0.01), (b) the frequency of SCE per cell (6.96 vs. 4.87, P<0.001), and (c) the percentage of HFC (19.9% vs. 4.1%, P<0.001). BD exposure had no significant effects on formation of micronuclei and on comet assay parameters. Effect of smoking was observed only for HFC in BD-exposed group. GSTM1 genotype affected chromosomal aberrations in exposed group, while GSTT1 genotype affected chromosomal aberrations in controls. No effect of GSTM1 or GSTT1 genotypes was observed on any other biomarkers used.     

Developmental regulation of the 3-methylcholanthrene- and dioxin-inducible CYP1A5 gene in chick embryo liver in vivo

Measurement of specific adducts to hemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in humans. The purpose of this study was to investigate the dose response for adduct formation and persistence in rats and mice during long-term low-level exposure to butadiene by inhalation. Adducts of 3,4-epoxy-1-butene, the primary metabolite of butadiene, with N-terminal valine in hemoglobin were determined in male B6C3F1 mice and male Sprague-Dawley rats following exposure to 0, 2, 10, or 100 ppm of 1,3-butadiene, 6 h/day, 5 days/week for 1, 2, 3, or 4 weeks. Blood samples were collected from groups of five mice and three rats at the end of each week during the 4 weeks of exposure and weekly for 3 weeks following the end of the 4-week exposure period. The increase and decrease, respectively, of the adduct levels during and following the end of the 4-week exposure followed closely the theoretical curve for adduct accumulation and removal for rats and mice, thereby demonstrating that the adducts are chemically stable in vivo and that the elimination follows the turnover of the red blood cells. The adduct level increased linearly with butadiene exposure concentration in the mice, whereas a deviation from linearity was observed in the rats. For example, after exposure to 100 ppm butadiene, the epoxybutene-hemoglobin adduct levels were about four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Blood concentrations of epoxybutene, estimated from hemoglobin adduct levels, were in general agreement with reported concentrations in mice and rats exposed by inhalation to 62.5 ppm. These studies show that adducts of epoxybutene with N-terminal valine in hemoglobin can be used to predict blood concentration of epoxybutene in experimental animals.     

Insulin and insulin-like growth factor-I signal transduction requires p21ras

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.     

Synthesis and in vitro cytotoxicity of 3-substituted-1,8-diazaanthraquinones produced by Lewis-acid catalyzed hetero Diels-Alder reaction

A hetero Diels-Alder reaction of quinoline-5,8-dione with 1-(N,N-dimethylamino)-3-methyl-1-aza-1,3-butadiene proceeded to give 3-methyl-1,8-diazaanthraquinone (100% regioselectivity) in the presence of Lewis-acid catalyst (ZnCl2 or ZnBr2). Subsequent functionalizations of the benzylic methyl group resulted in the 1,8-diazaanthraquinone analogues as potential antitumor agents. The most active compound, 8, exhibited in vitro cytotoxic activity comparable to that of doxorubicin.     

Monophosphate 32P-postlabeling assay of DNA adducts from 1,2:3,4-diepoxybutane, the most genotoxic metabolite of 1,3-butadiene: in vitro methodological studies and in vivo dosimetry

Among the main DNA-reactive metabolites of 1,3-butadiene (BD), both 1,2:3,4-butadiene diepoxide (BDE) and 1,2-epoxy-3-butene (BME) have been reported in mice and rats exposed to BD, but blood and tissue levels of these metabolites are much higher in mice than in rats under similar exposure conditions. BDE, being more reactive and genotoxic than BME, is thought to be responsible for the greater susceptibility of mice to BD carcinogenicity. While BDE is a DNA-alkylating agent and some BDE adducts have been characterized, no sufficiently sensitive method has been reported for studying BDE-DNA binding in vivo. In the present investigation, a modified dinucleotide/monophosphate version of the 32P-postlabeling assay was applied to detect BDE-DNA adducts, which were prepared by reacting BDE with calf thymus DNA or deoxyribooligonucleotides [(AC)10, (AG)10, (CCT)7 and (GGT)7] in vitro or with skin DNA of mice in vivo upon topical treatment. Optimal resolution by 2-D PEI-cellulose TLC of the highly polar 5'-monophosphate adducts was achieved at +4 degrees C using 0.3 M LiCI (DI) and 0.4 M NaCl, 0.04 M H3BO3, pH 7.6 (D2). The profiles of the 32P-postlabeled adducts were similar for calf thymus and skin DNA, with 3 major spots being detected. Adducts obtained in in vitro and in vivo experiments were compared by re- and cochromatography in 4 or 5 different solvents, and these experiments provided evidence that corresponding BDE adducts, for the most part, were identical and represented adenine derivatives. Guanine adducts were not detected by this method although literature data indicate their formation. Quantitatively, the assay responded linearly to adduct concentration, as shown in an experiment where BDE-modified skin DNA was serially diluted up to 81-fold with control DNA. The limit of detection was approximately 1 adduct in 10(8) normal nucleotides. Further, in an in vivo dosimetry study, skin DNA from groups of 8 individual mice treated with different doses of BDE (1.9, 5.7, 17, 51 and 153 mumol/mouse) for 3 days exhibited a linear relationship (r > or = 0.992) between adduct levels and dose. The results suggest that the 32P-postlabeling assay described herein will have utility in mechanistic studies and biomonitoring of DNA adduct formation from BDE and possibly other polar epoxides.     

High mutation frequency in ras genes of skin tumors isolated from DNA repair deficient xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) patients are clinically characterized by a very high incidence of skin cancers on exposed skin, at an early age. XP cells in vitro are strongly deficient in excision-repair and highly mutagenized by UV light. We were, therefore, interested in measuring mutation frequency and in determining mutation spectra in patients' tumors exposed to UV lesions. We chose to look at oncogene activation in skin tumors with the idea that more mutations, particularly of the ras gene family, would be found in XP tumors where lesions remain unrepaired compared to normal individuals. Our results clearly show that more than a 2-fold significantly higher mutation frequency (50%) of the ras genes was found in XP in contrast to control tumors (22%). The majority of the mutations were found at codon 12 of all three ras genes with a preponderance for N-ras in XP samples. The mutation spectra indicate that all mutations found were located opposite pyrimidine-pyrimidine sequences which represent a hot spot for UV-induced DNA lesions. Most of the mutations were of the type expected from studies performed in vitro with model systems. This high mutation frequency in XP was accompanied by a very high level of Ha-ras and c-myc gene amplification and rearrangement. All these data are consistent with a fundamental role of unrepaired UV-induced DNA lesions as an initiating event in human skin tumors on exposed parts of the body.     

A glutathione S-transferase with activity towards cis-1, 2-dichloroepoxyethane is involved in isoprene utilization by Rhodococcus sp. strain AD45

Rhodococcus sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene, cis-1, 2-dichloroethene to cis-1,2-dichloroepoxyethane, and trans-1, 2-dichloroethene to trans-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane and trans-1, 2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of cis-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3, 4-epoxy-3-methyl-1-butene, epoxypropane, cis-1,2-dichloroepoxyethane, and trans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1, 2-epoxyhexane was detected in cell extracts of cells exposed to 1, 2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH S-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.     

Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT)

Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial beta-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.     

Aggressive myeloma with subcutaneous tumor and pericardial involvement

We investigated the potential role of intercellular-adhesion molecule-1 (ICAM-1) in allergen-induced bronchial hyperresponsiveness (BHR) and inflammation in sensitised Brown-Norway rats. Rats were sensitised with ovalbumin (OA) intraperitoneally and 21 days later they were either exposed to 0.9% NaCl or 1% OA aerosol for 15 min. Rats exposed to OA aerosol were pretreated either with ICAM-1 antibody (3 mg/kg i.p. and i.v., 45 min prior to OA exposure) or with the diluent for the antibody. Eighteen to twenty-four hours after OA or 0.9% NaCl exposure, rats were anaesthetised, tracheostomised and mechanically ventilated, and airway responsiveness to acetylcholine (ACh) aerosol was measured as the provocative concentration of ACh needed to increase pulmorary resistance by 100% (PC100). Mean -log PC100 was increased in rats exposed to OA but pretreated with diluent (2.75 +/- 0.06) compared to rats treated with ICAM-1 antibody (2.51 +/- 0.08; < 0.05). However, only the former group showed significantly higher mean -log PC100 compared to the sensitised group exposed to 0.9% NaCl alone (2.22 +/- 0.12; p < 0.01). There was a significant increase in eosinophil and lymphocyte counts in bronchoalveolar lavage fluid at 24 h in rats pretreated with diluent compared to saline exposed rats. However, in ICAM-1 antibody-pretreated rats, eosinophil and lymphocyte counts were significantly different from diluent-treated ones. We conclude that ICAM-1 antibody inhibits BHR without reducing the influx of inflammatory cells.     

Angiogenesis correlates with vascular endothelial growth factor expression but not with Ki-ras oncogene activation in non-small cell lung carcinoma

A total of 195 non-small cell lung carcinoma (NSCLC) specimens were studied for the presence of mutations in their ras family genes, for tumor vascularity, and for their immunostaining pattern with an antibody to vascular endothelial growth factor (VEGF). ras mutation was found in 37 of 104 (34.6%) adenocarcinoma specimens, in 0 of 64 squamous cell carcinomas, and in 2 of 27 (7.4%) large cell undifferentiated carcinomas. All mutations were found on the Ki-ras gene, with 37 (95%) of them on codon 12 and the remaining 2 on codon 13. Thirty (77%) of the mutations were G to T transversions. There was a correlation between increasing tumor vascularity and VEGF immunostaining score, but there was no correlation between either of them with the activation of the ras oncogene. A study of VEGF mRNA expression in 14 NSCLC cell lines also demonstrated a lack of correlation between the constitutive expression levels of VEGF and the presence or absence of ras mutation in these cell lines. The results suggest that VEGF is a major angiogenesis factor in NSCLC but that other factors beside ras mutations may influence tumor vascularity in these tumors. The two parameters may potentially serve as independent prognostic factors in NSCLC.     

Exposure to toxic air contaminants in environmental tobacco smoke: an assessment for California based on personal monitoring data

The contribution of environmental tobacco smoke (ETS) to the exposure of adult nonsmoking Californians was determined for selected toxic air contaminants (TACs). The assessment was based on published measurements of ETS emission factors and personal exposures to volatile organic compounds. The human exposure studies were conducted in three California areas--Los Angeles, Pittsburgh/Antioch, and Woodland--between 1984 and 1990. We derived unexposed and passive population exposure distributions by randomly sampling the monitoring results for individuals classified according to exposure status (active smoker, passively exposed or unexposed to ETS during monitoring). The differences between the unexposed and passive distributions were used to estimate the ETS-only contribution for exposure to benzene, styrene, o-xylene, and m,p-xylene. Emission factors were then employed to infer the ETS-caused exposure to thirteen other compounds. The estimated arithmetic mean increments of 24-hour exposure attributable to ETS for the nonsmoking Californian population (age > or = 7) exposed to ETS are as follows (results in units of microgram m-3 exposure concentration; results using two different emission factors presented as a range): acetaldehyde 11-15; acetonitrile 7.0; acrylonitrile 0.49; benzene 1.02; 1,3-butadiene 0.75-2.3; 2-butanone 1.4; o-cresol 0.17; m,p-cresol 0.41; ethyl acrylate < 0.015; ethylbenzene 0.49-0.64; formaldehyde 6.5-8.2; n-nitrosodimethylamine 0.0028; phenol 1.4; styrene 0.36; toluene 3.1-3.2; o-xylene 0.77; m,p-xylene 0.99. The 90% confidence limits on these estimates due to the limited sample size in the studies are roughly x/ divided by 6. For four widely studied compounds, ETS is estimated to contribute the following percentages to the total inhalation exposure of all nonsmoking Californians: o-xylene 5%; m,p-xylene 3%; benzene 5%; and styrene 8%.     

Risk assessment of butadiene in ambient air; the approach used in the UK

The UK Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (COC) and the related Committee on Mutagenicity provided advice on 1,3-butadiene in 1992. This followed detailed consideration of the available mutagenicity, animal carcinogenicity and epidemiology data plus information on toxicokinetics. They concluded that 1,3-butadiene was an in vivo mutagen, a potent genotoxic animal carcinogen and should be regarded as a probable human carcinogen. The Department of Health is not aware of more recent data warranting reconsideration of these conclusions. General advice on setting air quality standards for carcinogenic air pollutants was given by the COC. Although the prudent assumption of the absence of any safe level for genotoxic carcinogens was preferred, a pragmatic approach based essentially on assessment of the exposure at which no increased risk would be detected, plus a safety factor, was considered reasonable for compounds like butadiene where exposure cannot be totally avoided. This approach, plus recognition that it is unadvisable to allow ambient levels of genotoxic carcinogens to rise, is used in the UK. The procedure by which the Department of Environment's Expert Panel on Air Quality Standards recommended a value of 1 ppb for butadiene based on these principles is described.     

Benign indomethacin-responsive headaches presenting in the orofacial region: eight case reports

High affinity antibodies were used for the quantitative assessment of the miscoding O4-ethylthymine (O4-EtThy) base lesion in nanogram amounts of membrane transblotted restriction fragments of ENU treated DNA. The polyclonal antibody (TB3) specifically recognized attomoles of the alkylation adducts in modified DNA with no cross-reactivity to an excess of unmodified DNA. The sensitivity of the immuno-quantitative method was determined to be in the range of 76 attomoles to 2.43 fmol, corresponding to 0.24 x 10(-7) to 7.9 x 10(-7) adducts per nucleotide in plasmid DNA. Modification levels in ras and tk genes were estimated as 0.025 and 0.014 adducts respectively. Specific antibody binding was proportional to the dose of ENU and size of the DNA fragments. In differentially ethylated ras gene, the amount of O4-EtThy was quantified as 0.026, 0.08 and 0.13 adducts per gene fragment. A DNA concentration dependent antibody binding was observed with large (23.13 and 9.41 kb) and smaller (2.02 kb) fragments of HindIII digested ENU treated phage lambda DNA. To monitor the repair of O4-EtThy lesions in specific segments, damage was assessed in sequences of plasmid DNA established in various Escherichia coli strains. The loss of antibody binding to O4-EtThy adducts in ethylated DNA fragments of 6.4 kb ras gene and 3.6 kb tk gene occurred with an approximate t1/2 of 45 and 35 min, respectively, in the repair proficient wild type E. coli. On the contrary, no repair was seen in the alkyltransferase deficient double mutant ada-ogt- strain. The results specifically demonstrate the sensitivity of the immunological technique and the unique ability of the O4-EtThy specific antibodies to scan this promutagenic base lesion and its repair in very small amounts of selected gene segments in DNA.     

Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin

The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.     

Physiological compensation in antisense transformants: specific induction of an "ersatz" glucan endo-1,3-beta-glucosidase in plants infected with necrotizing viruses

Plant class I glucan endo-1,3-beta-glucosidases (beta-1,3-glucanase; 1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) have been implicated in development and defense against pathogen attack. Nevertheless, beta-1,3-glucanase deficiencies generated by antisense transformation of Nicotiana sylvestris and tobacco have little biological effect. We report here that another beta-1,3-glucanase activity is induced in these deficient mutants after infection with necrotizing viruses. Induction of class I beta-1,3-glucanase was markedly inhibited in leaves of N. sylvestris and tobacco antisense transformants infected with tobacco necrosis virus and tobacco mosaic virus, respectively. A serologically distinct beta-1,3-glucanase activity was present in the infected antisense transformants but was absent in both healthy and infected control plants and in antisense transformants treated with the stress hormone ethylene. Immunoblot analyses, localization studies, and measurements of antibody specificity indicate that this compensatory beta-1,3-glucanase activity is an intracellular enzyme different from known tobacco beta-1,3-glucanases. Therefore, plants can compensate for a deficiency in enzyme activity by producing a functionally equivalent replacement--i.e., "ersatz"--protein or proteins. The fact that compensation for beta-1,3-glucanase activity occurs in response to infection argues strongly for an important role of these enzymes in pathogenesis.     

Effect of alcoholic intoxication on the appreciation of different types of humor.

Placed 36 male undergraduates into 1 of 3 intoxication conditions (no ethanol, low dose, high dose) and exposed them to humorous segments from TV programs. During manifest intoxication or the corresponding period in the no-intoxication (control) condition, they were exposed, in a balanced order, to a segment containing blunt (i.e., unsophisticated, raw) humor and to a segment containing subtle (i.e., sophisticated, refined) humor. Ss' facial reactions to the humorous stimuli were unobtrusively recorded and later analyzed by judges who were naive about the experimental conditions. Ss also rated the funniness of the segments. The perceived funniness of blunt humor was found to increase with ethanol intoxication. For subtle humor, in contrast, perceived funniness decreased with intoxication. This divergent interaction of reported humor appreciation was less apparent in the analysis of facial expressiveness. Although correlations between ratings and aspects of facial expression were generally positive, clearly corroborative support for the humor appraisal was found only in the frequency of smiles in response to subtle humor. (23 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)