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Polymorphic metabolism of certain chemical carcinogens may result in differences in susceptibility to cancers. Human CYP2E1 (cytochrome P450IIE1) is an enzyme involved in the metabolic activation of precarcinogens such as nitrosamines. We detected a restriction fragment length polymorphism (RFLP) of the human CYP2E1 gene for the restriction endonuclease Dra I. The distribution of this polymorphism was examined among lung cancer patients (n = 91), patients with cancer of the digestive tract (n = 45) and controls (n = 76). A significant difference in the distribution was observed between lung cancer patients and controls (chi 2 = 11.4 with 2 df; p < 0.005). On the other hand, there was no significant difference between patients between cancer of the digestive tract and controls (chi 2 = 4.87 with 2 df; NS). This finding suggests that the Dra I polymorphism of the CYP2E1 gene is associated with susceptibility to lung cancer. In addition, an association was found between the amount of lifelong smoking exposure and the distribution of the genotypes of the RFLP among lung cancer patients. The distribution pattern seemed deviated from that of controls especially in the population of low smoking exposure. Our Northern blot analysis data using RNA from human liver autopsy samples suggest that the Dra I polymorphism might be associated with the gene expression of CYP2E1 at mRNA level.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

Cytochrome P-450MC was induced in pulmonary microsomes of 3-methylcholanthrene-treated rats and also at low level in that of isosafrole-treated rats. Cytochrome P-450d was not detected in the lungs of 3-methylcholanthrene- or isosafrole-treated rats by the method of Western blot analysis with a polyclonal antibody raised against cytochrome P-450c which is equally effective against P-450d, nor by the method of Northern hybridization probed with pcP450mc3 (P-450d probe). Complementary DNA of P-450MC was isolated from rat pulmonary cDNA library and the nucleotide sequence of pulmonary cDNA was compared with that of hepatic P-450c cDNA reported by Yabusaki et al. There was no gross change in nucleotide sequences of cDNA between pulmonary P-450MC and hepatic P-450c.

Two major forms of hepatic cytochrome P-450 (hepatic P-450MCI and P-450MCII) were purified approximately 5-fold from liver microsomes in Syrian golden hamsters treated with 3-methylcholanthrene (MC). The purified preparations of hepatic P-450MCI and P-450MCII contained 9.6 and 8.3 nmol cytochrome P-450 (P-450) per mg protein, respectively, and were essentially free from NADPH-cytochrome c (P-450) reductase (fpT), NADH-cytochrome b5 reductase and cytochrome b5. By sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of hepatic P-450MCI and P-450MCII were estimated to be 56,000 and 53,500. Further, a major form of pulmonary P-450 (P-450MC) were purified from lung microsomes of MC-treated hamster, and contained 14.2 nmol P-450 per mg protein, and estimated to be 56,000 in monomeric molecular weight, indicating the similar molecular weight to hepatic P-450MCI in the hamster. From the absorption spectra the oxidized forms of hepatic P-450MCI and P-450MCII were high- and low-spin ferric hemoproteins, respectively, and pulmonary P-450MC was similar to hepatic P-450MCII in their hemoprotein spin state. No difference, however, was observed in the CO-reduced forms among hepatic P-450MCI, P-450MCII and pulmonary P-450MC, all exhibiting 446.5 nm Soret bands. In a reconstituted system containing fpT and dilauroylphosphatidylcholine (DLPC), pulmonary P-450MC efficiently catalyzed benzo[a]pyrene (BP) hydroxylation at a rate of 11.4 mol formed per min per mol P-450, but hepatic P-450MCI and P-450MCII both exhibited lower levels, e.g., 0.49 and 0.54, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 313 fold from lung microsomes of 3-methylcholanthrene (MC)-treated rats. The purified preparation contained 12.5 nmol of cytochrome P-450 per mg protein and was essentially free from NADPH-cytochrome c-reductase, NADH-cytochrome b5-reductase, and cytochrome b5. By SDS-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 54,000, which is smaller than that of a major form of hepatic cytochrome P-450 (hepatic P-450MC) purified from MC-treated rats. The peptide patterns of the purified pulmonary P-450MC were apparently different from those of hepatic P-450MC on partial proteolysis with either S. aureus V8 protease or papain, indicating that the primary structure of pulmonary P-450MC is different from that of hepatic P-450MC. In reconstituted systems, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation, but showed a low activity for benzphetamine N-demethylation. Pulmonary P-450MC formed a single precipitation line with the antibody prepared against hepatic P-450MC in Ouchterlony double diffusion analysis. The pulmonary and hepatic P-450MC activities of benzo(a)pyrene hydroxylation and ethoxycoumarin O-deethylation were inhibited in the same manner by the antibody against hepatic P-450MC. These results suggest that pulmonary P-450MC is immunologically very similar to hepatic P-450MC.