Evaluation of the effect of 5-aminolevulinic acid hexyl ester (H-ALA) PDT on EBV LMP1 protein expression in human nasopharyngeal cells.

Nasopharyngeal carcinoma (NPC) is of high prevalence in Hong Kong and southern China. The pathogenesis of NPC is closely associated with Epstein-Barr virus (EBV) infection via regulation of viral oncoprotein latent membrane protein 1 (LMP1). The conventional treatment for NPC is chemo-radiotherapy, but the prognosis remains poor for advanced stage, recurrent and metastatic NPC. Photodynamic therapy (PDT) is a therapeutic approach to combat tumors. PDT effectiveness depends on the interaction of photosensitizers, light and molecular oxygen. 5- aminolevulinic acid hexyl derivative (H-ALA) is one of the photosensitizers derived from 5-ALA. H-ALA with improved lipophilic properties by adding a long lipophilic chain (hexyl group) to 5-ALA, resulted in better penetration into cell cytoplasm. In this study, the effect of H-ALA-PDT on NPC cells (EBV positive C666-1 and EBV negative CNE2) was investigated. The H-ALA mediated cellular uptake and cytotoxicity was revealed via flow cytometry analysis and MTT assay respectively. H-ALA PDT mediated protein modulation was analysed by western blot analysis. Our finding reported that the cellular uptake of H-ALA in C666-1 and CNE2 cells was in a time dependent manner. H-ALA PDT was effective to C666-1 and CNE2 cells. EBV LMP1 proteins was expressed in C666-1 cells only and its expression was responsive to H-ALA PDT in a dose dependent manner. This work revealed the potential of H-ALA PDT as a treatment regiment for EBV positive NPC cells. Understanding the mechanism of H-ALA mediated PDT could develop improved strategies for the treatment of NPC.

Photodynamic effects on nasopharyngeal carcinoma (NPC) cells with 5-aminolevulinic acid or its hexyl ester.

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Hong Kong and southern China. To explore a new modality of NPC treatment, 5-aminolevulinic acid (ALA) or its hexyl ester (ALA-H) mediated photodynamic therapy (PDT) was studied in vitro. The results show that NPC cells are sensitive to both ALA and ALA-H mediated PDT. However, ALA-H PDT is much more effective at cell inactivation than ALA-PDT, due to a higher efficiency of ALA-H on producing endogenous protoporphyrin (PpIX) in cells. Both apoptosis and necrosis are involved in cell death, but apoptosis plays a major role under the short time incubation of drugs. ALA and ALA-H mediated PDT not only destroy the cells directly, but also inhibit the expression of matrix metalloproteinase-2 (MMP2) in cells, a maker for tumor metastasis. The ALA-H shows promising PDT results on NPC in vitro; therefore it is worth investigating further in vivo for NPC treatment.

The cytotoxic and genotoxic potential of 5-aminolevulinic acid on lymphocytes: a comet assay study.

BACKGROUND: 5-aminolevulinic acid (ALA) and its hexylester (ALA-H) are the drugs currently used in photodynamic therapy (PDT). The side effect, especially the long-term side effect of these drugs is a problem of concern in this field, which has not been clearly understood yet. PURPOSE: The normal lymphocytes and nasopharyngeal carcinoma (NPC) cells were used as the cell models to evaluate the side effects of ALA or ALA-H in the absence of light or under sub-lethal doses of light. METHODS: The cytotoxic and DNA-damaging effects of ALA or ALA-H on lymphocytes and NPC cells were studied by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the alkaline comet assay. ALA at 0.75 mM concentration and ALA-H at 10-microM concentrations were selected in the studies. This is because under these concentrations, ALA- or ALA-H-mediated PDT can destroy most NPC cells in vitro. The intracellular distributions of the protoporphyrin IX (PpIX), induced by the ALA or ALA-H, were measured by the confocal laser scanning microscope to provide more information for understanding the DNA damage. RESULTS: The incubation of 0.75 mM ALA or 10 microM ALA-H alone (without light) did not cause DNA damage as well as the considerable cytotoxic effect on NPC cells. However, after ALA (0.75 mM) incubation and without light irradiation, the serious cytotoxicity and remarkable DNA damage were found in lymphocytes. When the lymphocytes were incubated with ALA-H (10 microM) alone (in the absence of light), no DNA damage could be detected and a slight cytotoxic effect was found. Both ALA and ALA-H induced PpIX in the lymphocytes. The fluorescence images of PpIX intracellular localization demonstrated that the PpIX diffused into the nuclear region in ALA-(0.75 mM)-incubated lymphocytes but not existed in the nucleus of ALA-H(10 microM)- incubated lymphocytes, providing an explanation for the facts that ALA (0.75 mM) induced the DNA damage while ALA-H (10 microM) did not. CONCLUSION: These results suggested that the genotoxic potential of lymphocytes seems high for ALA (0.75 mM) and could be excluded for ALA-H (10 microM).

Minimization of heterocyclic amines and thermal inactivation of Escherichia coli in fried ground beef.

BACKGROUND: Heterocyclic amine carcinogens are formed during the cooking of a number of foods, especially well-done meats. Lower temperatures and shorter cooking times can minimize the formation of these carcinogens, yet a major food safety concern is that pathogens in the meat must be thermally inactivated. This study investigated cooking techniques that minimize heterocyclic amine formation while simultaneously destroying contaminating bacteria. METHODS: Ground beef patties were inoculated with Escherichia coli K12 bacteria and fried to internal temperatures ranging from 35 degrees C to 70 degrees C in a skillet preheated to 160 degrees C, 180 degrees C, or 200 degrees C. Each patty was then analyzed for four common heterocyclic amines and for surviving bacteria. Additionally, the frequency of turning of the beef patty during cooking was varied (a single turn or multiple turns), length of time required for each patty to reach 70 degrees C was recorded, and heterocyclic amine levels were determined. An additional pan temperature of 250 degrees C was tested for its effect on heterocyclic amine formation but not on bacterial killing. Statistical tests were two-sided. RESULTS: Colony-forming bacteria were reduced by five orders of magnitude at internal temperatures greater than 60 degrees C, regardless of cooking method, and were completely inactivated at 70 degrees C. For patties turned just once, heterocyclic amine levels increased as the cooking temperatures increased. However, levels of heterocyclic amines were statistically significantly lower with turning every minute. For each pan temperature, patties reached 70 degrees C internal temperature sooner when they were turned every minute than when they were turned just once during cooking. CONCLUSION: Lowering the pan temperature and turning the patties frequently can greatly reduce the formation of heterocyclic amines and can simultaneously achieve bacterial inactivation with little or no increase in cooking time, ensuring a product that is safe for human consumption.

Effect of FosPeg® mediated photoactivation on P-gp/ABCB1 protein expression in human nasopharyngeal carcinoma cells.

Multidrug resistance (MDR) refers to the ability of cancer cells to develop cross resistance to a range of anticancer drugs which are structurally and functionally unrelated. P-glycoprotein (P-gp) is the best studied MDR phenotype in photodynamic therapy (PDT) treated cells. Our pervious study demonstrated that FosPeg® mediated PDT is effective to NPC cell line models. In this in vitro study, the expression of MDR1 gene and its product P-gp in undifferentiated, poorly differentiated and well differentiated human nasopharyngeal carcinoma (NPC) cells were investigated. The influence of P-gp efflux activities on photosensitizer FosPeg® was also examined. Regardless of the differentiation status, PDT tested NPC cell lines all expressed P-gp protein. Results indicated that FosPeg® photoactivation could heighten the expression of MDR1 gene and P-gp transporter protein in a dose dependent manner. Up to 2-fold increase of P-gp protein expression were seen in NPC cells after FosPeg® mediated PDT. Interestingly, our finding demonstrated that FosPeg® mediated PDT efficiency is independent to the MDR1 gene and P-gp protein expression in NPC cells. FosPeg® itself is not the substrate of P-gp transporter protein and no efflux of FosPeg® were observed in NPC cells. Therefore, the PDT efficiency would not be affected even though FosPeg® mediated PDT could induce MDR1 gene and P-gp protein expression in NPC cells. FosPeg® mediated PDT could be a potential therapeutic approach for MDR cancer patients.

Specificity of base substitution mutations induced by the dietary carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) 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 PhlP and IQ in Salmonella typhimurium strains TA100 and TA1535 were determined by colony hybridization analysis. Both PhlP and IQ induced predominantly GC-->TA transversions in strain TA100 (rfa,delta uvrB/pKM101) with a pronounced preference for the second codon position (CCC--> CAC; 72% of total). PhlP also reverted strain TA1535 (rfa, delta uvrB) efficiently at concentrations similar to those required for strain TA100. In contrast to the PhlP-induced mutational profile observed in strain TA100, in strain TA1535 PhlP induced exclusively GC-->AT transitions at the second codon position (CCC-->CTC; 96-99% of total). Base substitution mutagenesis induced by heterocyclic amines related to PhlP 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 PhlP- guanosine adduct at the C-8 position. The GC-->AT transition mutations induced by PhlP in strain TA1535 appear to be SOS-independent, however, suggesting that these mutations may arise from the formation of PhlP-DNA adducts other than the replication-blocking C8-dG lesion.

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 D(50) 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.

Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.

Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.

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.

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.

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.

Implications of prolonged fetal heart rate deceleration during the second stage of labor.

In order to evaluate the neonatal outcomes of infants who had prolonged fetal heart rate (FHR) deceleration during the second stage of labor, the neonatal outcomes of 24 infants born after vaginal delivery at 37 to 42 weeks of gestation with prolonged FHR deceleration during the second stage of labor were compared with the outcomes of 28 infants of a similar gestational age who had normal FHR patterns. No differences in the Apgar scores, mean umbilical PaCO2, PaO2, HCO3- or base deficit values were observed between the two groups, but the mean pH values differed significantly. The occurrences of a 1-minute Apgar score < 7, umbilical arterial pH < 7.20, presence of meconium and admission to the neonatal intensive care unit were higher in the study group, but were not significantly different. None of the 24 infants with prolonged FHR deceleration experienced birth trauma, meconium aspiration, neonatal seizure or neonatal death, but three were found to have congenital heart disease. We conclude that prolonged FHR deceleration during the second stage of labor without FHR abnormalities during the first stage of labor is not always associated with an adverse neonatal outcome and does not mandate the need for surgical or immediate vaginal delivery. Their appearance on FHR tracings requires the implementation of additional methods to assess fetal well-being and also to diagnose fetal distress.

Photodynamic therapy (PDT) - Initiation of apoptosis via activation of stress-activated p38 MAPK and JNK signal pathway in H460 cell lines.

AIMS: The purpose of this study was to investigate the photoefficacies of protoporphyrin IX (PpIX) generated by drug precursor 5-aminolevulinic acid (ALA) and its hexyl ester (H-ALA) on two human non-small lung carcinoma cell lines (H460/Bcl-2 and H460/neo). MAIN METHODS: Drug uptake and the photoefficacies of PpIX were measured by flow cytometry and MTT assay; while the mode of cell death and alternation of signal transduction pathways were studied with 4',6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis, respectively. KEY FINDINGS: The flow cytometric analysis of H-ALA (5µM) uptake revealed optimal fluorescent intensity at 8h incubation, while ALA (0.5mM) was still far from saturation. The LD(30) of H-ALA-PDT was 30µM, 2J/cm(2), while the LD(30) of ALA-PDT was 3mM, 2J/cm(2). The dark toxicities mediated by both pro-drug H-ALA and ALA were negligible. By DAPI staining, apoptotic cell death was observed. In addition, by Western blot analysis, H-ALA- and ALA-mediated PDT initiated apoptotic cell death via the up-regulation and activation of p38 mitogen activated protein kinase (MAPK), the stress-activated c-jun N-terminal kinases (JNK) and ERK. SIGNIFICANCE: These results suggested that H-ALA and ALA mediated PDT displayed similar photocytotoxicities towards the two non-small lung cancer cells. Our present study also demonstrates H-ALA or ALA mediated PDT in H460 cells are closely related to the activation of p38 MAPK and JNK signalling pathway.

Introduction of cytochrome P450IA2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency.

We introduced into the CHO cell line the cDNA of the mouse cytochrome P3450 (P450IA2) gene, which oxidizes aromatic amines. A cDNA clone of P3450 was transfected into mutant UV5 cells, which is defective in nucleotide excision repair. Expression of the P3450 cDNA was measured using 9000 x g supernatant (S9) fractions from CHO cells to evaluate Salmonella TA1538 mutagenicity with the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The P3450-expressing clone UV5P3 was reverted to repair proficiency using ethyl methanesulfonate to obtain the UV-resistant clone 5P3R2, which maintained the same level of P3450 protein activity as UV5P3. These genetically similar cell lines were compared for toxicity and mutation induction at the aprt locus. With 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (the most prevalent mutagen found in fried beef) the differential sensitivity due to repair deficiency/proficiency was approximately 40-fold, and with IQ there were smaller, but significant, differences in sensitivity. These genotoxic effects occurred at doses that were approximately 10 times lower than those that previously gave similar effects in S9-mediated assays. Thus, these cell lines should be valuable for genotoxicity analysis as well as important for assessing DNA repair when evaluating compounds that undergo metabolic activation.

Electrocardiographic changes in amitriptyline poisoning.

In imipramine and amitriptyline poisoning the electrocardiographic abnormalities comprise arrhythmias, widening of the QRS complex, and marked changes in die S-T segment. These features were found to be of value in the differential diagnosis of unknown poisoning. The unusual configuration of qR with raised S-T segment in V1, simulating myocardial infarction, was seen in one of our patients and has been noted in four cases reported by other workers.

Identification of aprt gene mutations induced in repair-deficient and P450-expressing CHO cells by the food-related mutagen/carcinogen, PhIP.

We investigated the specific sequence changes produced by the dietary mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in UV5P3 cells [a Chinese hamster ovary (CHO) cell line]. Sequence analysis of the PhIP-induced mutations in the adenine phosphoribosyltransferase (aprt) gene, which is heterozygous in the UV5P3 cells, can provide insight into the mutagenic mechanism in these repair-deficient cells expressing P4501A2. Two allele-specific 20 mer oligonucleotide primer pairs were used in the polymerase chain reaction and the allele of interest was amplified. Single-base transversions occurred in 31/32 PhIP-induced mutants; of these, 6 were A.T-->T.A, 18 were C.G-->A.T and 6 were G.C-->T.A. Twenty of the 30 changes altered specific amino acid sequences and the other 10 resulted in a stop codon. On mutant had a change from C.G-->G.C at the 3' splice site of intron 4, thereby creating a new AG splice acceptor site. Another mutant had an insertion of T within a run of repeated sequences and resulted in a frameshift mutation. There were three 'hot-spots', two at the 3' end of exon 2 and one at the beginning of exon 3; 6 (19%) mutants showed a change from A.T-->T.A (exon 2, amino acid residue 57), 11 (34%) mutants from C.G-->A.T (exon 2, amino acid residue 62), and 7 (22%) mutants from C.G-->A.T (exon 3, amino acid residue 66). Consequently, 75% of the mutations were observed at these three sites. In contrast, none of the 20 spontaneous mutants had alterations at these hotspot sites. The mutations induced by PhIP in these repair-deficient CHO cells were unique and specific, and suggest that these sequences, if found in important genes controlling cell replication and survival, may be more susceptible to mutation from these food mutagens than genes not containing these sequences.

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

A correlation of Salmonella mutagenicity with DNA adducts induced by the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The correlation of bacterial mutagenicity with DNA adducts from the heterocyclic amine cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated in Salmonella typhimurium strains TA98 (uvrB deficient) and TA1978 (uvrB proficient). Bacterial cells were exposed to PhIP using a modification of the Ames/Salmonella microsuspension assay. Half of the cells, generated from a 90 min pre-incubation and washing, were plated for revertant formation while the remaining half was subjected to DNA adduct analysis via 32P-postlabeling. In TA98, DNA adducts were detected at an RAL (relative adduct labeling) of 10 x 10(-7) and 21 x 10(-7) at PhIP concentrations of 5.5 and 17 microM, respectively. This corresponded to 28.8 and 20.9 adducts/revertant, respectively. These values were based on the assumption that only four repeating GC bases within a 75 DNA base region is the gene target site for PhIP induced mutations. In TA1978, no revertants above background were detected at any concentration of PhIP tested. DNA adducts, however, were detected at 11 x 10(-7) and 21 x 10(-7) adducts per nucleotide at 223 and 1116 microM PhIP, respectively. The lack of detectable revertants, but the presence of DNA adducts, suggests pre-mutational lesions did occur during the 90 min pre-incubation. Presumably, when the S9 activating system and PhIP were removed (via washing with phosphate buffered saline) prior to plating, the cells containing an intact uvrB repair system repaired the lesions during the incubation time on the plates. In conclusion, the induction of revertants by adducts appears quite efficient, as approximately 25 adducts are required for one mutational event in the excision repair deficient bacteria.