Edward Bresnick

Northern hybridization analysis of RNA using diethylpyrocarbonate-treated nonfat milk.

A simple and relatively inexpensive method for hybridizing RNA that is immobilized on nitrocellulose to a radioactive DNA probe is presented. The procedure, a modification of the Bovine Lacto Transfer Technique Optimizer method of Johnson et al. (1984) Gene Anal. Tech. 1, 3-8, uses nonfat milk which has been treated with the RNase inhibitor, diethylpyrocarbonate (DEPC), to saturate nonspecific binding sites on nitrocellulose paper, and to wash off unbound radioactivity. The DEPC-nonfat milk hybridization technique is faster, less costly, and more reliable than standard Northern blot hybridization conditions, yielding sharp, clear bands of RNA on autoradiographs with virtually no background reactivity.

Further characterization of the polycyclic aromatic hydrocarbon binding properties of the 4S protein

A 4-S protein which specifically binds [3H]benzo(a)pyrene and other polycyclic aromatic hydrocarbons has been investigated in the rat using a hydroxylapatite assay and sucrose gradient analysis. Although there was significant interanimal variation, the specific polycyclic aromatic hydrocarbon binding activity appeared to be highest in 4-week-old male rats and declined with age. The specific [3H]benzo(a)pyrene binding activity was induced after pretreatment with either phenobarbital or isosafrole as evidenced by a 72 and 61% increase, respectively, over untreated controls. No apparent increase in specific binding activity was observed after pretreatment of animals with 3-methylcholanthrene. Pretreatment with either phenobarbital or isosafrole also resulted in the appearance of a small, nonspecific, benzo(a)pyrene binding peak at the 8- to 9-S region in the sucrose density gradients. This 8-S peak was not seen in untreated control animals and represented low affinity, high capacity binding sites. In contrast to the 8-S protein, the 4-S binding protein had low affinity for polychlorinated aromatic compounds such as tetrachlorodibenzodioxin and tetrachlorodibenzofuran. The addition of a 200-fold excess of tetrachlorodibenzofuran to incubations did not displace [3H]benzo(a)pyrene from the 4-S protein. The addition of sodium molybdate to isolation buffers, known to stabilize certain hormone receptors, did not alter the sedimentation coefficient or the specific binding activity of the 4-S protein. These experiments indicate that the 4-S protein does not appear to be a subunit of the 8-S protein. We conclude that in the rat the 4-S protein is distinct from the 8-S protein and the 4-S species may regulate the polycyclic aromatic hydrocarbon-induced expression of aryl hydrocarbon hydroxylase activity.

The effect of dietary fat on metastasis of the lewis lung carcinoma and the BALB/c mammary carcinoma

The effect of feeding mice diets high in beef tallow (high in saturated fat) or corn oil (high in polyunsaturated fat) on the production of lung metastases by the Lewis lung carcinoma and the BALB/c mammary tumor was determined. Diets were fed ad libitum, and the mice fed the high-fat (24.6%) diets consumed more calories and gained more weight than those fed the control (5%) diets. With the Lewis lung carcinoma, we found that both high-fat diets significantly increased the growth of the primary tumor in the footpad as well as the number of spontaneous metastases produced after the primary was removed; this was in comparison with results from the appropriate control diets. With the BALB/c mammary tumor, the high-fat beef tallow diet (but not the corn oil diet) significantly increased the number of lung metastases formed after tail vein injection. In addition, the group given the control corn oil diet had more metastases than the group given the control beef tallow diet. Overall, these studies showed that the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of low-fat diets. However, the results varied depending on the tumor model used and the type of fat.

3-Methylcholanthrene-induced expression of the cytochrome P-450c gene

Transcriptional control of 3-methylcholanthrene-dependent cytochrome P-450c nuclear RNA induction was directly observed in an in vitro rat liver nuclear transcription system. Mercurated and radiolabeled ribonucleotides were incorporated into nuclear RNA transcribed in vitro, which was then isolated using thiopropyl-Sepharose 6B affinity chromatography. Dot hybridization experiments were carried out using bacteriophage M13 subclones of pRSA57 (a cDNA clone for rat serum albumin), pEB339 (a cDNA clone for rat cytochrome P-450c), and clone 46 (a cDNA clone for mouse cytochrome P1-450). The results of these studies demonstrate that 3-methylcholanthrene does not significantly influence the transcription of the rat serum albumin gene, but does increase the transcription of the cytochrome P-450c gene. Nuclear RNA precursors to the cytochrome P-450c mRNA were characterized by Northern blot analysis. Clone 46 hybridized to nuclear RNA species of 6.7 and 4.0 kb, in addition to the 3.0-kb cytochrome P-450c mRNA. pA8 (a genomic clone for rat cytochrome P-450c), hybridized to the same nuclear RNA species in addition to nuclear RNA species of 4.3, 3.4, and 2.2 kb. M13pd15 (a genomic clone containing information for the first intron of the cytochrome P-450c gene) hybridized to nuclear RNA species of 6.7 and 4.3 kb. All of these nuclear RNA species are polyadenylated. The mRNA coding for cytochrome P-450c was induced maximally in hepatic nuclei at 3 h following 3-methylcholanthrene administration. Maximal accumulation of cytochrome P-450c mRNA in hepatic cytosol has been previously shown to occur at approximately 15 h following 3-methylcholanthrene administration (Bresnick, E., Brosseau, M., Levin, W., Reik, L., Ryan, D. E., and Thomas, P. E. (1981) Proc. Natl. Acad. Sci. USA 78, 4083-4087). These data implicate a possible role of nuclear RNA transport in the regulation of induction of cytochrome P-450c, although further investigations are indicated.

The 4S binding protein acts as a trans-regulator of the polycyclic hydrocarbon-inducible cytochrome P450

A model has been proposed for the induction of cytochrome P450c in liver by polycyclic hydrocarbons such as benzo(a)pyrene (BaP) and 3-methylcholanthrene (3MC). The polycyclic hydrocarbon interacts in specific, saturable, and high-affinity fashion with a rat liver cytosolic 4s binding protein. The latter enters the nucleus, complexes to 5′ upstream regions of the cytochrome P450c gene, and stimulates the transcription. The 4s binding protein has been purified from rat liver and its substrate specificity has been determined. The affinity for 3MC or BaP is 1–2 mM. The binding protein has been demonstrated to complex with specific 5′-upstream regions of the P450c gene by using a filtration assay as well as exonuclease footprinting. In addition, the binding protein stimulates in vitro transcription with upstream regions of the P450c gene as template; these data confirm the hypotheses.

Synergy of phenobarbital and 3-methylcholanthrene in “superinduction” of cytochrome P-450c mRNA but not enzyme activity

The combination of phenobarbital and 3-methylcholanthrene in the inductive process of the rat hepatic cytochrome P-450c gene was evaluated. Daily injections of phenobarbital (80 mg/kg, i.p.) had little or no effect on the amount of poly (A)+ RNA encoding cytochrome P-450c, whereas a single injection of 3-methylcholanthrene (25 mg/kg, i.p.) produced a significant accumulation at 15 hr in cytosolic mRNA coding for cytochrome P-450c. Four daily injections of phenobarbital followed by a single dose of 3-methylcholanthrene produced 5-24 times more poly (A)+ RNA coding for P-450c than 3-methylcholanthrene treatment alone. This superinduction of RNA transcripts was also observed for a species coding for cytochrome P-450d, which was increased 3-6 times over 3-methylcholanthrene treatment alone. However, the elevated concentration of transcripts for both the P-450d and P-450c RNA species did not result in an increase in the marker enzyme activity for cytochrome P-450c, 7-ethoxyresorufin O-deethylase. These data implicate a regulatory step in the induction of cytochrome P-450c enzyme activity which must occur at a level beyond transcription.

Structural details of the human cytochrome P-450c gene

Aryl hydrocarbon hydroxylase activity is most closely associated with cytochrome P-450c in the rat and cytochrome P1-450 in the mouse. The sequence for the orthologous human gene coding for this enzymatic activity has been determined from several sources: cytochrome P-450c isolated from human embryonic DNA [K. Kawajiri, J. Watanabe, O. Gotoh, Y. Tagashiri, K. Sogawa, and Y. Fujii-Kuriyama (1986) Eur. J. Biochem. 139, 219-225], human lymphocytes in our own laboratory, and cytochrome P1-450 isolated from the established human breast carcinoma cell line, MCF-7 [A.K. Jaiswal, F. J. Gonzalez, and D. W. Nebert (1985) Nucleic Acids Res. 13, 4503-4520]. The data from our laboratory agree well with the sequence derived from human embryonic DNA, but differs significantly from that reported for the gene isolated from MCF-7 cells. Among these differences are a 320-bp insert and a 650-bp deletion in intron 1 relative to the sequence derived from the established cell line. We observe two mRNA species that hybridize to cytochrome P-450c probes, one expected at 2.7 kb and an additional 2.0-kb species. Finally, we note additional hybridization bands in 11% of the population examined by Southern blot analysis, representing either a second rare allele or, more likely, a duplication of at least a portion of the cytochrome P-450c gene.

6-Nitrobenzo[a]pyrene can be denitrated during mammalian metabolism.

Nitropolycyclic aromatic hydrocarbons (nitroarenes), including 6-nitrobenzo[a]pyrene (6-NBap), occur in our environment and are mutagenic in bacterial mutagenesis assays. The mutagenicity of 6-NBaP is enhanced when rat liver S9 is added. To investigate the cause of this increased activity, the metabolism of 6-NBaP was carried out with a total rat liver homogenate obtained from 3-methylcholanthrene- (MC-) induced rats, a 9000 X g supernatant enzyme, and with both unwashed and washed microsomes. Ring-hydroxylated 6-NBaP was detected. On the basis of retention times for known standards in a high-performance liquid chromatographic system, benzo[a]pyrene (BaP) and 6-acetoxy-BaP (6-OAcBaP) were isolated as products. BaP was further characterized via ultraviolet (UV) and mass spectra and 6-OAcBaP by UV, mass, and nuclear magnetic resonance (NMR) spectra. 6-HydroxyBaP (6-OHBaP) was also detected by UV and mass spectra. It is suggested that BaP is formed via a nitroanion radical of 6-NBaP and undergoes metabolism, while the 6-OHBaP is acetylated to form 6-OAcBaP. The acetyl donor remains to be identified.

Metabolism of 6-nitrobenzo[a]pyrene in rat lung preparations

6-Nitrobenzo[alpha]pyrene (6-NBaP) occurs in our environment. Since human exposure to environmental contaminants may occur via the inhalation route, we examined the metabolites of 6-NBaP formed in lung preparations, and compared the metabolite profile to that which was found with liver. The metabolites formed in both liver and lung preparations consisted of ring-hydroxylated 6-NBaP, 6-hydroxybenzo[a]pyrene (6-OHBaP) and small amounts of benzo[a]pyrene. In the lung experiments, 6-OHBaP most frequently oxidized to quinones. The mechanism for the formation of 6-OHBaP from 6-NBaP remains to be elucidated.

Nuclease hypersensitivity of the rat cytochrome P450IA1 gene

The bovine pancreatic deoxyribonuclease I (DNAase I) hypersensitivity of the rat cytochrome P450IA1 gene was investigated. A nuclease-hypersensitive region was observed at approximately 3.2 to 5.1 kilobase pairs upstream of exon 1 in adult and fetal rat liver. This region did not necessarily correlate with gene expression following 3-methylcholanthrene induction, although it may determine the potential for inducibility of this gene.