John C. Loper

Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.

Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: identification of a new P450 gene family.

The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures are discussed for their possible role in the inducibility of this gene. Expression of the P450alk gene was achieved in Saccharomyces cerevisiae using the yeast alcohol dehydrogenase expression system after removal of the P450alk gene flanking regions. The resultant expressed protein had a molecular mass slightly greater than that of P450alk from C. tropicalis. This alteration did not prevent the function and the localization of P450alk expressed in S. cerevisiae, as this organism showed an acquired microsome-bound activity for the terminal hydroxylation of lauric acid. The deduced P450alk amino acid sequence was compared with members of the nine known P450 gene families. These comparisons indicated that P450alk had a low relationship with these members and was therefore the first member (A1) of a new P450 gene family (LII).

Isolation of a cytochrome P-450 structural gene from Saccharomyces cerevisiae

We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14 alpha-demethylase. Two plasmids were isolated which transformed yeast to both increased resistance to Kc and increased levels of total P-450. Hybrid-selection and immunoprecipitation experiments showed that these plasmids, pVK1 and pVK2, contained the structural gene for an S. cerevisiae P-450. This conclusion was confirmed by the nucleotide sequence of a portion of pVK2, which revealed an open reading frame encoding a characteristic P-450 heme-binding region.

Disruption of the Saccharomyces Cerevlsiae gene for NADPH-cytochrome P450 reductase causes increased sensitivity to ketoconazole

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14 alpha-demethylase. Resistance is restored through complementation by the plasmid-borne wild type gene from either S. cerevisiae or Candida tropicalis. Neither Southern hybridization nor Western immunoblot techniques provided evidence for a second NADPH-cytochrome P450 reductase gene, suggesting that an alternate pathway may provide for the functions of this reductase in S. cerevisiae.

Non-mutagenicity for salmonella of the chlorinated hydrocarbons aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone

A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-trichlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlorobenzene were shown to differ from S9 of either control or Aroclor 1254-induced rats in the capacity to activate 2-aminoanthracene mutagenesis.

Cytochrome P450 oxidoreductase gene and its differentially terminated cDNAs from the white rot fungus Phanerochaete chrysosporium.

Abstract The white rot fungus Phanerochaete chrysosporium metabolizes a range of xenobiotics via P450 mono-oxygenation, particularly under peroxidase-suppressing culture conditions. Here we report the cloning and analysis of the gene from this fungus for the cytochrome P450 oxidoreductase (CPR) and its differentially terminated cDNAs. Using a PCR-based approach with degenerate primers, a 285-bp genomic fragment was isolated from the two widely studied strains BKM-F 1767 and ME 446, and was identified as a CPR gene segment based on sequence comparison with the database. A clone containing the full-length CPR gene was isolated from a BKM-F 1767 genomic library using the PCR-generated segment as a probe, and the 3937-bp insert was sequenced by gene walking. Based on the detection of conserved CPR motifs, a coding region of 2381 bp was identified with a 991-bp segment 5′ to the putative ATG start codon. Two cDNAs with differentially terminated transcripts were isolated and sequenced. Comparison of the gene and the cDNA sequences confirmed the presence of three introns (62 bp, 50 bp, and 58 bp). Sequence identity and a phylogenetic comparison of the deduced protein (736 aa) with other CPRs in the database suggested that P. chrysosporium CPR is the largest CPR known and is more closely related to animal (36–38%) and yeast (37–38%) CPRs than to plant CPRs (33–35%). The availability of this gene will facilitate further studies on understanding the potent xenobiotic mono-oxygenation systems in this model white rot fungus.

Tandem cytochrome P450 monooxygenase genes and splice variants in the white rot fungus Phanerochaete chrysosporium: cloning, sequence analysis, and regulation of differential expression

Phanerochaete chrysosporium, the model white rot fungus, uses P450 monooxygenation for oxidation of xenobiotic chemicals. To characterize its P450 systems, we have isolated two tandemly linked full-length P450 monooxygenase genes, pc-1 (2510bp) and pc-2 (2503bp). cDNA sequence and splice site analysis showed multiple small putative introns (35-82bp), 17 in pc-1 and 16 in pc-2. The deduced P450 proteins PC-1 (531 aa, MW 60,070) and PC-2 (538 aa, MW 61,765) showed 55% aa identity with each other and low relatedness (25-36%) with P450 gene families CYP52, CYP86, CYP4, and CYP3 decreasing in that order. Alternate splicing was identified for pc-1 transcription. Real-time quantitative RT-PCR analysis on 4-day cultures showed an elevated level of expression ( approximately 8-fold) of pc-1 in nutrient-limited (ligninolytic) as compared to nutrient-rich (non-ligninolytic) conditions. Interestingly, the pc-1 gene is expressible in both primary and secondary phases of growth with peak expression occurring with secondary metabolism.

Multiple P450alk (cytochrome P450 alkane hydroxylase) genes from the halotolerant yeast Debaryomyces hansenii

The halotolerant alkane-assimilating yeast Debaryomyces hansenii was examined for P450 alkane hydroxylase genes known to be required for alkane assimilation in Candida. Four distinct P450alk gene segments and an allelic segment were isolated using PCR based on degenerate primers derived from the CYP52 family of alkane-inducible P450 genes. A screen of a genomic library (15-20kb inserts) constructed for this study, using a probe based on the PCR-isolated segments, yielded seven clones. This has led to the isolation and sequence of two full-length genes DH-ALK1 and DH-ALK2. These genes, each with an ORF of 1557 bp (519 aa), contained no apparent introns and showed 64% nucleotide sequence homology (61% based on the deduced amino acid sequences). The deduced proteins had predicted molecular weights of 59,254Da (DH-ALK1) and 59,614Da (DH-ALK2) and have been designated CYP52A12 and CYP52A13 by the P450 Nomenclature Committee. Phylogenetic analysis based on Neighbor Joining Tree showed that DH-ALK1 and DH-ALK2 constitute new genes located on two distinct branches and are most related to the gene CYP52A3 (60% deduced aa homology) and are least related to the gene CYP52C2 (41% deduced aa homology), both of C. maltosa. The isolated genes will provide tools to better understand the diversity of the P450alk family in eukaryotic microorganisms adapted to varied environmental conditions.

Separation of Mutagens from Drinking Water using Coupled Bioassay/Analytical Fractionation

Abstract A general preparative procedure has been developed for the isolation of mutagenic components from organic residues of drinking water. This procedure features the Salmonella-microsome mutagenicity assay coupled with an analytical fractionation method which progressively focuses to the bioactive constituents of the complex mixture. The three-step method consists of: 1) semi-solid/liquid extraction; 2) radially compressed column octadecyl-silane reverse phase HPLC; 3) SEP-PAKR concentration and solvent exchange of HPLC subfractions for chemical and biological characterization. Using as a model a carbon chloroform extracted organic residue mixture prepared from drinking water several years ago, three mutagenic HPLC subfractions were isolated, accounting for the bulk of the mutagenic activity of this complex mixture. Preliminary GC/MS results indicate the mutagenic activity is due to isomeric chlorinated aliphatic ethers. The biological characterization suggests the mutagenic specific activity of thes...

Analytical isolation, separation and identification of mutagens from nonvolatile organics of drinking water.

A general procedure has been developed for the concentration/fractionation of mutagenic residue organics from small, less than 50L, and large, to 1200L, volumes of drinking water obtained from a variety of sources. This procedure features concentration of the residue organics chromatographically by passage of the water through XAD-2 and XAD-7 resins in specially designed columns, details of which are given. The residue organics are eluted from the resins via organic solvents, followed by solvent removal and subsequent bioassay for mutagenicity. Then the residue organics are fractionated via a coupled bioassay/analytical fractionation method which progressively focuses to the bioactive constituents of the complex mixture of residue organics. In this report, results for the optimal operation and validation of the concentration system are given, using drinking water derived from an industrially polluted river system, a wilderness river system and a major aquifer system. The predominant type of mutagenesis observed for the residue organics isolated from these samples was direct-acting to the Salmonella tester strain, TA98, which was decreased by the addition of the metabolic activation system from the livers of rats previously treated with Arochlor 1254. Some TA100 direct-acting mutagenesis was observed for all samples. Fractionation of the residue organics indicated the mutagens to be nonpolar. Samples of residue organics collected over a period of a year from each type of drinking water showed no discernable pattern of mutagenesis versus season. The methodologies described in this paper provide a comprehensive approach for the concentration/isolation of residue organics from drinking water for studies to identify biohazardous compounds and to characterize these compounds biologically.