Rebekah W. Wu

Health risks of heterocyclic amines.

Common cooking procedures such as broiling, frying, barbecuing (flame-grilling), heat processing and pyrolysis of protein-rich foods induce the formation of potent mutagenic and carcinogenic heterocyclic amines. These same compounds produce tumors at multiple organ sites in both mice and rats. One example of these induced tumors has also been seen in nonhuman primates. Risk assessment for the human population consuming these compounds requires the integration of knowledge of dosimetry, metabolism, carcinogenic potency, and epidemiology. When this integration is done in even a preliminary way as is done here, the range of risk for an individual from these compounds is enormous. Exposure contributes a range of 200-fold or more and metabolism and DNA repair differences among individuals could easily be an additional 10-fold between individuals. This indicates that differences in human cancer risk for heterocyclic amines could range more than a thousandfold between individuals based on exposure and genetic susceptibility.

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.

Differential effect of acetyltransferase expression on the genotoxicity of heterocyclic amines in CHO cells

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase (aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed approximately 1000-fold increase in sensitivity to the killing effect of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.

Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine

To test the hypothesis that the sulfotransferase gene plays a role in the phase II bioactivation of PhIP, a heterocyclic amine found in cooked meats, we transfected the UV5P3 cell line with cDNA plasmids of human aryl sulfotransferases (HAST1 and HAST3). UV5P3 is a nucleotide excision repair‐deficient and P4501A2‐expressing CHO cell line that we have previously developed. Functionally transformed clones were identified by the differential cytotoxicity (DC) assay that used PhIP as the cytotoxic agent. Two clones designated 5P3H1 and 5P3H3, expressing HAST1 and HAST3, respectively, were chosen for further characterization. Correct fragment sizes of the sulfotransferase cDNAs were identified in both cell lines by polymerase chain reaction. Immunoblot analysis confirmed the expression of the sulfotransferase proteins. The addition of the sulfotransferase inhibitor DCNP decreased the cytotoxic effects of PhIP in a dose‐dependent manner. The increase in cell growth was 6.5‐fold for 5P3H1 and 2.4‐fold for 5P3H3, relative to values obtained without DCNP. Based on D50 values, the dose that reduced the survival to 50% relative to untreated controls, the cytotoxic effect of PhIP was increased threefold for 5P3H1 and 1.87‐fold for 5P3H3 cell lines over the parental UV5P3 line. There was also a small increase in the mutation response at the aprt locus. These newly established 5P3H1 and 5P3H3 sulfotransferase‐expressing cells provide valuable mechanistic information of the bioactivation of PhIP and related compounds. Environ. Mol. Mutagen. 35:57–65, 2000. Published 2000 Wiley‐Liss, Inc.

Genetically modified Chinese hamster ovary (CHO) cells for studying the genotoxicity of heterocyclic amines from cooked foods

We have developed metabolically competent Chinese hamster ovary (CHO) cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) but not with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines.

Methylcobalamin methylation of chloroplatinate: bound chloride, valence state, and relative mutagenicity

Abstract A methyl‐Pt compound formed by incubating K2PtCl6 with methylcobalamin (MeB‐12) is further characterized with respect to its chloride content, its Pt oxidation state, and its electro‐phoretic mobility. The complexed chloride has been determined from nuclear magnetic resonance (NMR) spectra of the Cl displaced by competing thiocyanate (SCN ) anions. Release of 3 chlorides per Pt combined with a Me/Pt ratio of 1.0 indicates a metal coordination number of 4. This is in agreement with X‐ray photoelectron spectra (ESCA) which show that the majority of the Me‐Pt product contains Pt in the +2 valence state. Based on this data and the anionic nature of the product at pHs from 2.5–7.5 it 2‐is presently represented as MePtCl3. In comparison to the closely related complexes K2PtCl4 and K2PtCl6, MePtCl3 ‐2 is slightly less cytotoxic to an auxotrdphic Chinese hamster ovary cell line (CHO AUXB1). All three of these chloro‐Pt compounds at concentrations of 15–70 μ? induce a 6–10 fold increase in the frequency o...

Platinum tetrachloride: Mutagenicity and methylation with methylcobálamin

It was reported earlier that methylcobalamin (MeB‐12) plus K2PtCl6 or Na2PtCl6 under acidic conditions yields a single square planar Pt2+ species, MePtCl2‐ 3. A reaction between MeB‐12 and PtCl4 in...

Specificity of base substitution mutations induced by the dietary carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Salmonella

The base pair substitution mutational profiles induced by the heterocyclic amine cooked food mutagens PhIP and IQ in Salmonella typhimurium strains TA100 and TA1535 were determined by colony hybridization analysis. Both PhIP and IQ induced predominantly G→TA transversions in strain TA100 (rfa,ΔuvrB/pKM101) with a pronounced preference for the second codon position (CC→CAC; 72% of total). PhIP also reverted strain TA1535 (rfa, ΔuvrB) efficiently at concentrations similar to those required for strain TA100. In contrast to the PhIP‐induced mutational profile observed in strain TA100, in strain TA1535 PhIP induced exclusively G→AT transitions at the second codon position (CC→CTC; 96–99% of total). Base substitution mutagenesis induced by heterocyclic amines related to PhIP is generally SOS‐dependent, requiring the presence of plasmid pKM101 in Salmonella hisG46 strains. Thus, the SOS dependent reversion of S. typhimurium strain TA100 probably reflects error‐prone lesion bypass at the major PhIP‐ guanosine adduct at the C‐8 position. The G→AT transition mutations induced by PhIP in strain TA1535 appear to be SOS‐independent, however, suggesting that these mutations may arise from the formation of PhIP‐DNA adducts other than the replication‐blocking C8‐dG lesion. Environ. Mol. Mutagen. 31:327–332, 1998

A Method to Detect Differential Gene Expression in Cross-Species Hybridization Experiments at Gene and Probe Level

Motivation Whole genome microarrays are increasingly becoming the method of choice to study responses in model organisms to disease, stressors or other stimuli. However, whole genome sequences are available for only some model organisms, and there are still many species whose genome sequences are not yet available. Cross-species studies, where arrays developed for one species are used to study gene expression in a closely related species, have been used to address this gap, with some promising results. Current analytical methods have included filtration of some probes or genes that showed low hybridization activities. But consensus filtration schemes are still not available. Results A novel masking procedure is proposed based on currently available target species sequences to filter out probes and study a cross-species data set using this masking procedure and gene-set analysis. Gene-set analysis evaluates the association of some priori defined gene groups with a phenotype of interest. Two methods, Gene Set Enrichment Analysis (GSEA) and Test of Test Statistics (ToTS) were investigated. The results showed that masking procedure together with ToTS method worked well in our data set. The results from an alternative way to study cross-species hybridization experiments without masking are also presented. We hypothesize that the multi-probes structure of Affymetrix microarrays makes it possible to aggregate the effects of both well-hybridized and poorly-hybridized probes to study a group of genes. The principles of gene-set analysis were applied to the probe-level data instead of gene-level data. The results showed that ToTS can give valuable information and thus can be used as a powerful technique for analyzing cross-species hybridization experiments. Availability Software in the form of R code is available at http://anson.ucdavis.edu/~ychen/cross-species.html Supplementary Data Supplementary data are available at http://anson.ucdavis.edu/~ychen/cross-species.html

Induced reversion of a Chinese hamster ovary triple auxotroph validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.