A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

The sporicidal activity of ozone in the gas phase was investigated. Spores of six strains of Bacillus species deposited on filter paper or glass fibre filter were conditioned at different relative humidities (r.h.), and then exposed to ozone ranging in concentration from 0.5 to 3.0 mg/l at different r.h. There was a lag phase in the initial stage of exposure followed by an exponential decrease in the number of survivors with time, although no lag phase was observed with one strain. Inactivation rates increased with increasing exposure r.h. while no significant inactivation was attained at a r.h. of 50% or below. The conditioning r.h. influenced the duration of the lag phase. The D-values (decimal reduction time) in the logarithmic phase varied roughly in inverse proportion to the ozone concentration.

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Generation of free radicals in the reaction of ozone with blood samples and related salt solutions was investigated in vitro by using ESR spin-trapping technique with DMPO. In the reactions of low levels of ozone, a carbon-centered radical was spin-trapped with DMPO, giving rise to the 6-line ESR signal in both whole blood and blood plasma. In the blood plasma, DMPO-spin adduct of hydroxyl radical (DMPO-OH) was detected together with the spin adduct of carbon-centered radical. The present spin-trapping study demonstrates that, when exposed to ozone, 0.9% NaCl solution in the presence of DMPO gives rise to the formation of DMPO-OH. The addition effects of ethanol, which is a .OH scavenger, into the NaCl solution reveal that DMPO-OH is produced by the reaction of DMPO with both .OH and unidentified oxidants originated from the reaction of Cl- and ozone. Based on these observations, we consider that .OH is generated similarly in the blood plasma exposed to ozone. The ESR study of DMPO-spin adducts in the ozone-exposed aqueous solution of NaOCl indicates that Cl- reacts with ozone to give ClO-. Presumably, further oxidation of ClO- by ozone leads to the formations of .OH and the unidentified oxidants.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.