CYP3A5 and NAT2 gene polymorphisms: role in childhood acute lymphoblastic leukemia risk and treatment outcome.

Susceptibility to acute lymphoblastic leukemia can be highly influenced by genetic polymorphisms in metabolizing enzyme genes of environmental carcinogens. This study aimed to evaluate the impact of the CYP3A5 and NAT2 metabolizing enzyme polymorphisms on the risk of childhood acute lymphoblastic leukemia. The analysis was conducted on 204 ALL patients and in 364 controls from a Brazilian population, using PCR-RFLP. The CYP3A5 3 polymorphic homozygous genotype was more frequent among ALL patients and the 3 allele variant was significantly associated with increased risk of childhood ALL (OR = 0.29; 95% CI, 0.14-0.60). The homozygous polymorphic genotype for the 6 allele variant was extremely rare and found in only two individuals. The heterozygous frequencies were similar for the ALL group and the control group. No significant differences were observed between the groups analyzed regarding NAT2 variant polymorphisms. None of the polymorphisms analyzed was related to treatment outcome. The results suggest that CYP3A5 3 polymorphism may play an important role in the risk of childhood ALL.

Liver-selective expression of human arylamine N-acetyltransferase NAT2 in transgenic mice.

Human arylamine N-acetyltransferase 2 (NAT2) mediates the biotransformation of arylamine drugs and procarcinogens into either innocuous or reactive DNA-damaging metabolites and is expressed predominantly in liver. Interspecies differences and incongruous results between in vitro, in vivo, and epidemiological studies make it difficult to extrapolate animal results to human risk. We have generated human NAT2 transgenic mice on both C57BL/6 (hNAT2(tg)) and Nat1/2 null backgrounds [hNAT2(tg)Nat1/2(-/-)], in which liver-selective expression of human NAT2 is driven by the mouse albumin promoter. We detected expression of the human NAT2 transcript and protein in mouse liver by real-time PCR and Western blot analysis. NAT2 enzyme activity, measured using the human NAT2-selective substrate sulfamethazine (SMZ), was 40- to 80-fold higher in liver cytosols from hNAT2(tg)Nat1/2(-/-) mice than in wild-type mice. An unexpected gender difference was observed, with males displaying 2-fold higher activity than females. Transgenic mice also had an increased in vivo plasma clearance of SMZ and higher levels of N-acetylated SMZ than wild-type mice. Liver expression of human NAT2 did not affect the disposition of the human NAT1-selective substrate p-aminosalicylic acid (PAS), because hNAT2(tg)Nat1/2(-/-) mice displayed in vivo PAS pharmacokinetic profiles similar to those of Nat1/2(-/-) mice. The metabolism of 4-aminobiphenyl was similar between hNAT2(tg)Nat1/2(-/-) and wild-type mice with the exception of a more liver-restricted pattern in hNAT2(tg)Nat1/2(-/-) mice and lower activity in females. Overall, the hNAT2(tg)Nat1/2(-/-) mouse mimics human expression of NAT2 and may thus be of value in clarifying the role of human NAT2 in arylamine clearance, detoxification, and bioactivation.

Genetic polymorphisms of NAT2 and risk of acute myeloid leukemia: A case-control study.

Our purpose was to investigate the possible associations between N-acetyltransferase-2 (NAT2) gene polymorphisms and the risk of acute myeloid leukemia (AML) in Chinese Han population.A case-control study was conducted including 98 AML cases and 112 healthy controls. NAT2 gene 2 polymorphisms rs1799930 and rs1799931 were genotyped using direct sequencing. Chi-square test was performed to compare the genotype and allele distribution differences between groups. Odds ratio (OR) with 95% confidence interval (CI) was calculated to estimate the association between NAT2 gene polymorphisms and AML onset.A remarkable decrease trend of rs1799931 GA genotype was detected in AML patients compared with controls, whereas the ancestral GG genotype frequency increased in cases (P < .05). And the mutant A allele of rs1799931 significantly reduced the risk of AML by 0.585-fold versus the ancestral G allele carriers (OR = 0.585, 95% CI = 0.361-0.950). But the distributions of rs1799930 genotype and allele were similar between groups (P > .05).Our findings suggested that NAT2 gene polymorphism rs1799931 was associated with decreased risk of AML and was likely to be a protective factor against AML development.

Competitive allele-specific short oligonucleotide hybridization (CASSOH) with enzyme-linked immunosorbent assay (ELISA) for the detection of pharmacogenetic single nucleotide polymorphisms (SNPs).

Individualization of drug therapy through genetic testing would maximize the effectiveness of medication and minimize its risks. Recent progress in genetic testing technologies has been remarkable, and they have been applied for the analysis of genetic polymorphisms that regulate drug responses. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We previously reported a novel DNA diagnostic method for detecting single nucleotide polymorphisms (SNPs) using competitive allele-specific short oligonucleotide hybridization (CASSOH) with an immunochromatographic strip. We have developed the method further in order to incorporate an enzyme-linked immunosorbent assay (ELISA) into the final detection step; this enables multiple SNP detection. Special ELISA chips have been fabricated so that disposal of buffer waste is not required and handling procedures are minimized. This method (CASSOH-ELISA) has been successfully applied for the detection of clinically important SNPs in drug metabolism, such as N-acetyltransferase 2, NAT2*6 (590G>A) and NAT*7 (857G>A), and mitochondrial DNA (1555A>G). It would also facilitate point-of-care genetic testing for potentially diverse clinical applications.

Rapid identification of the NAT2 genotype in tuberculosis patients by multicolor melting curve analysis.

AIM: NAT2 genotype is an indicator for isoniazid dosage adjusting for tuberculosis treatment. Multicolor melting curve analysis (MMCA) was evaluated as a potential method for NAT2 genotyping. MATERIALS & METHODS: 352 blood samples were analyzed by MMCA kit (Zeesan Biotech Co., Xiamen, China) targeting NAT2 SNPs at T341C, C481T, G590A and G857A, and direct sequencing was used as control. RESULTS: The sensitivity, specificity and accuracy of the MMCA assay for rapid NAT2 genotype detection were 97.9, 99.6 and 99.1% respectively, whereas for intermediate genotypes the values were 99.5, 98.7 and 99.1%, respectively, and for slow genotypes the values were 100% for the three aspects. The 24 saliva and blood for the control samples were also successfully analyzed using the MMCA assay, both produced uniform outcomes. CONCLUSION: The MMCA assay described in our study is very promising for the efficient determination of NAT2 genotype, and can facilitate the personalized dosing of isoniazid.

Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance.

It has been historically assumed that the pineal gland is the major source of melatonin (N-acetyl-5-methoxytryptamine) in vertebrates. Melatonin plays a central role in fine-tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin-N-acetyltransferase, and hydroxyindole-O-methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin-stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL-2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance.

Effects of aspirin on the in vitro and in vivo acetylation of 2-aminofluorene in Sprague-Dawley rats.

The present study was undertaken to determine the effect of acetylsalicylic acid acid on the in vitro N-acetyltransferase (NAT) enzyme activity and in vivo acetylation of 2-aminofluorene in laboratory rats. In the in vitro experiments, cytosols of blood, bladder, colon and liver cells, with or without acetylsalicylic acid co-treatment, showed different percentages of 2-aminofluorene acetylation. The data indicated that there was decreased NAT activity associated with increased acetylsalicylic acid in the cytosol reaction. In the in vitro experiments, values of apparent Km and Vmax decreased by 4% and 21%, respectively, for acetylation of AF in blood NAT, 28% and 31% for acetylation of AF in bladder NAT, 12% and 25% for acetylation of AF in colon NAT, and 50% and 35% for acetylation of AF in liver NAT. In the in vivo experiments, pretreatment with acetylsalicylic acid (50 mg/kg) 48 h prior to the administration of 2-aminofluorene (50 mg/kg) resulted in 24% and 28% decreases in the fecal and urinary recovery of N-acetyl-2-aminofluorene and a 26% decrease in the metabolic clearance of 2-aminofluorene to N-acetyl-2-aminofluorene. This is the first demonstration of acetylsalicylic acid (Aspirin) inhibition of arylamine N-acetyltransferase activity showing decreases in the N-acetylation of carcinogens in vivo.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice.

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Genotyping of N-acetylation polymorphism and correlation with procainamide metabolism.

We studied the genotypes of polymorphic N-acetyltransferase (NAT2) in 145 Japanese subjects by the polymerase chain reaction-restriction fragment length polymorphism method. The rapid-type NAT2*4 was expressed at a higher frequency (68.6%) than the slow-type genes with specific point mutations (NAT2*6A, 19.3%; NAT2*7B, 9.7%; NAT2*5B, 2.4%). The frequency of NAT2* genotypes consisted of 44% of a homozygote of NAT2*4, 49% of a heterozygote of NAT2*4 and mutant genes, and 7% of a combination of mutant genes. The metabolic activity for procainamide to N-acetylprocainamide was measured in 11 healthy subjects whose genotype had been determined. Although the acetylation activity substantially varied interindividually, the variability was considerably reduced after classification according to the genotype. The N-acetylprocainamide/procainamide ratio in urinary excretion was 0.60 +/- 0.17 (mean +/- SD) for those with NAT2*4/*4, 0.37 +/- 0.06 for NAT2*4/*6A, 0.40 +/- 0.03 for NAT2*4/*7B, and 0.17 for NAT2*6A/*7B. The results indicated that the NAT2* genotype correlates with acetylation of procainamide.

Quality of NAT2 genotyping with restriction fragment length polymorphism using DNA isolated from frozen urine.

In large studies and under field conditions common to epidemiological research, factors outside of and inside the laboratory can introduce misclassification of genetic susceptibility markers. Few reports have been made on the accuracy of genotyping individuals using DNA extracted from frozen urine that was stored for approximately 20 years. This study was performed to determine the reproducibility and accuracy of N-acetyltransferase 2 (NAT2) genotyping by RFLP analysis using DNA from stored urine. To obtain long-term frozen urine and blood samples from the same person, the databases of two large prospective studies were linked by name and date of birth. Six polymorphisms within the coding region of NAT2 were determined in 65 urine and blood samples after which, genotypes and imputed phenotypes (rapid, slow) were derived. To test reproducibility, all of the six polymorphisms were determined twice in 47 urine-blood pairs. Reproducibility of imputed phenotypes was 91.5% in urine samples and 97.9% in blood samples. To test accuracy, results for all six polymorphisms were compared between urine and blood DNA. All of the kappa's were at least 0.85 except one. Identical results for all six polymorphisms were seen in 78.5% of urine-blood pairs. Taking blood samples as a reference standard, rapid acetylators were classified as rapid in 97% of subjects (95% confidence interval, 90-100%), and slow acetylators were classified as slow also in 97% of subjects (95% confidence interval, 91-100%), when using urine. This study shows that stored urine samples can be used for DNA genotyping in large cohort studies, when blood samples are not available.

Inter-individual variation of human blood N-acetyltransferase activity in vitro.

Inter-individual variation in the in vitro acetylation of the antibacterial drug sulphamethazine by human whole blood was studied using reverse phase HPLC. The mean (range) values of blood N-acetyltransferase activity in vitro were 0.50 (0.29-0.83) nmol per 10(9) red blood cells (rbc) (N = 23), 3.33 (2.22-5.27) nmol per 10(9) rbc (N = 27) and 9.36 (6.72-15.76) nmol per 10(9) rbc (N = 23) at initial sulphamethazine concentrations of 0.018 mM, 0.18 mM and 1.44 mM respectively. The mean (range) values of the initial rate of sulphamethazine acetylation at these substrate concentrations were 28.1 (20.9-35.0) pmol/hr per 10(9) rbc (N = 11), 0.26 (0.18-0.42) nmol/hr per 10(9) rbc (N = 19) and 0.91 (0.61-1.50) nmol/hr per 10(9) rbc (N = 14) respectively. The mean (range) half life of thermal inactivation of blood acetylation capacity at 50 degrees was 0.91 (0.59-1.27) min (N = 12) at an initial substrate concentration of 0.18 mM. In each of these cases, there was no significant differences between the values obtained using blood samples from rapid and slow acetylators. Intra-individual variation of blood N-acetyltransferase activity was studied in a single subject on 24 separate occasions during a two year period and was less than 10% at each of the three sulphamethazine concentrations studied. The correlation between the in vitro blood N-acetyltransferase activity of eight volunteers measured on two separate occasions at least 6 weeks apart was 0.84, 0.98 and 0.98 at initial sulphamethazine concentrations of 0.018 mM, 0.18 mM and 1.44 mM respectively. Increasing the acetyl-CoA concentration of blood samples from 4 subjects by 0.34, 0.85 and 1.67 mM significantly increased both the initial acetylation rate of sulphamethazine and the amount of acetylsulphamethazine produced after an incubation time of 24 hr (initial sulphamethazine concentration = 0.18 mM).

Arylamine N-acetyltransferase in human red blood cells.

N-Acetyltransferase activities associated with erythrocytes from 20 individuals have been determined with p-aminobenzoic acid as substrate. A three-fold variation in Vmax is found. The N-acetyltransferase genotype of the individuals has been determined and there is no correlation between the extent of acetylation measured in the individuals' erythrocytes and the inheritance of alleles at the polymorphic NAT locus. Folate is confirmed to be an inhibitor of arylamine N-acetyltransferase activity measured in erythrocytes. The content of folate in erythrocytes of individuals also varies. The individual with the maximum folate content has the minimum N-acetyltransferase activity. The monomorphic N-acetyltransferase gene from individuals spanning the range of N-acetyltransferase activity have been amplified, using the polymerase chain reaction. The pattern of restriction enzyme digestion of the monomorphic N-acetyltransferase gene with a series of eight restriction enzymes is the same for individuals spanning the activity range of arylamine N-acetyltransferase in their erythrocytes.

In vitro studies on the deacetylation-reacetylation of arylamides and the transacetylation of arylamines by human and rat whole blood.

Human and rat whole blood were shown to metabolize the aromatic amides acetanilide and phenacetin by deacetylation followed by reacetylation in vitro. Derivatives of the parent compounds labelled with deuterium in the N-acetyl group produced non-labelled material after incubation. The reaction was monitored by capillary gas chromatographic-mass spectrometric (GC-MS) analysis. There was no significant difference in the acetyl group exchange of these substrates using blood samples donated by non-diabetic volunteers or Type 2 diabetic patients (respective mean +/- SEM values = 4.0 +/- 0.2% and 4.2 +/- 0.3% for trideuteroacetanilide, 6.2 +/- 0.6% and 6.1 +/- 0.3% for trideuterophenacetin). Increasing the glucose concentration in the incubation medium by 50 mmol/L significantly (P less than 0.01) increased deacetylation-reacetylation of trideuteroacetanilide in each group (4.6 +/- 0.2% and 4.7 +/- 0.2% for non-diabetic and diabetic subjects, respectively). In rat blood the amount of deacetylation-reacetylation was much higher: 7.2 +/- 0.6% and 8.3 +/- 0.7% for trideuteroacetanilide and trideuterophenacetin, respectively. Induction of experimental diabetes using streptozotocin did not significantly change the extent of deacetylation-reacetylation of either deuterated substrate (10.1 +/- 2.1% and 9.5 +/- 1.1%). Elevation of the incubation glucose concentration by 50 mmol/L produced an increase in acetyl group exchange (for trideuteroacetanilide) in diabetic (14.3 +/- 2.2%) and non-diabetic (10.6 +/- 1.0%) rats. The donation of acetyl groups (transacetylation) was observed after incubation of blood samples from both diabetic and non-diabetic human subjects and rats with trideuterophenacetin and a molar excess of aniline. This reaction significantly (P less than 0.001) decreased the acetyl group exchange of trideuterophenacetin (these values were 4.5 +/- 0.4% and 3.4 +/- 0.6% using samples from non-diabetic human subjects and rats, respectively) and demonstrated the ability of whole blood to catalyse transacetylation (acetyl-CoA-independent acetylation). There was correlation between the amount of (unlabelled) acetanilide produced by acetylation with acetyl-CoA and the percentage present as trideuteroacetanilide. The proportion of trideuteroacetanilide was higher using rat blood (e.g. the values for non-diabetic subjects were 25.5 +/- 1.7% vs 8.5 +/- 0.3%; P less than 0.001) although the total amount of acetanilide produced was lower (0.54 +/- 0.14 nmol vs 1.82 +/- 0.23 nmol; P less than 0.05) than that observed using human blood.

The effect of streptozotocin-induced diabetes on the in vivo acetylation capacity and the in vitro blood N-acetyltransferase activity of the adult male Sprague-Dawley rat.

Induction of experimental diabetes using streptozotocin significantly reduced the extent of sulphamethazine acetylation by Sprague-Dawley rats. This treatment did not significantly change the total amount of sulphonomide excreted in the urine. The in vitro blood N-acetyltransferase activity of rats treated with streptozotocin was significantly higher than that of untreated animals. Increasing the in vitro glucose concentration of blood samples from both groups significantly increased the amount of acetylsulphamethazine produced.

Immunomodulation and drug acetylation: influence of the immunomodulator tilorone on hepatic, renal and blood N-acetyltransferase activity and on hepatic cytosolic acetyl coenzyme A content.

The biochemical alteration responsible for immunomodulator enhancement of drug acetylation in vivo was probed ex vivo and in vitro in the rat. Rat liver or kidney cytosol, obtained by differential centrifugation, or whole blood served as the source of N-acetyltransferase (NAT). Addition of tilorone (0.5-8.0 mM) to incubation mixtures containing procainamide (PA, 0.6 mM) and acetyl coenzyme A (AcCoA, 0.42 mM) resulted in the inhibition of N-acetylprocainamide formation, while lower concentrations of tilorone had no effect. Pretreatment of rats with tilorone (50 mg/kg) administered orally 48 hr prior to sacrifice did not alter hepatic apparent Km and Vmax for NAT toward PA compared to control animals. Utilization of an AcCoA regenerating system in the incubation mixtures also resulted in no significant differences in the apparent Michaelis-Menten parameters obtained. Acetylation activity in kidney and whole blood also was not altered by immunomodulator pretreatment. Hepatic cytosolic AcCoA content was reduced significantly 48 hr after tilorone pretreatment (5.10 +/- 2.1 vs 11.97 +/- 2.2 nmol/mg protein) (P less than 0.05). These data indicate that an increase in NAT content or activity is not the biochemical alteration responsible for immunomodulator enhancement of drug acetylation, and that the required cofactor, cytosolic AcCoA, is decreased by immunomodulator pretreatment.

Caffeine metabolism and the risk of spontaneous abortion of normal karyotype fetuses.

OBJECTIVE: To investigate whether the rate of caffeine metabolism influences spontaneous abortion risk. METHODS: We studied 101 women with normal karyotype spontaneous abortions and 953 pregnant women at 6-12 gestational weeks. Participants reported on caffeine intake and provided urine for phenotyping cytochrome P4501A2 (CYP1A2) activity and blood for genotyping N-acetylation (NAT2) status. We calculated odds ratios (OR) and 95% confidence intervals (CI) to evaluate the association between each of the two metabolic indices and spontaneous abortion risk and also the potential interaction between caffeine intake and metabolic activity on such risk. In calculating the associations between the metabolic indices and risk of spontaneous abortion, we had 80% power to detect an OR of 2.1, with a Type I error of 0.05. RESULTS: Slow acetylators had a nonsignificantly increased risk for spontaneous abortion (OR 1.36, 95% CI 0.84, 2.21) and recurrent spontaneous abortion (OR 2.51, 95% CI 0.81, 7.76). In contrast, low CYP1A2 activity was associated with a significantly decreased risk for spontaneous abortion (OR 0.35, 95% CI 0.20, 0.63). Caffeine was a risk factor for spontaneous abortion among women with high, but not low, CYP1A2 activity (OR 2.42, 95% CI 1.01, 5.80 for 100-299 mg/day; OR 3.17, 95% CI 1.22, 8.22 for 300 mg/day or more, among women with high CYP1A2 activity). CONCLUSION: The findings indicate that high CYP1A2 activity may increase the risk of spontaneous abortion, independently or by modifying the effect of caffeine. The results regarding NAT2 are less conclusive but suggest that slow acetylators may be at elevated risk of spontaneous abortion.

Effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the acetylation of 2-aminofluorene and DNA-2-aminofluorene adducts in the rat.

The effects of the synthetic phenolic antioxidants (butylated hydroxyanisole and butylated hydroxytoluene) on the in vivo acetylation of 2-aminofluorene and formation of DNA-2-aminofluorene adducts were investigated in male Sprague-Dawley rats. For in vitro examination, cytosols and intact cells, with or without butylated hydroxyanisole and butylated hydroxytoluene co-treatment, showed different percentages of 2-aminofluorene acetylation and DNA-2-aminofluorene adducts. For in vivo examination, pre-treatment of male rats with butylated hydroxyanisole and butylated hydroxytoluene (10 mg/kg) 48 h prior to the administration of 2-aminofluorene (50 mg/kg) resulted in 34% and 18%, 29% and 20% decreases, respectively, in the urinary and fecal recovery of N-acetyl-2-aminofluorene, and 34% and 19% decreases, respectively, in the metabolic clearance of 2-aminofluorene to N-acetyl-2-aminofluorene. Following exposure of rats to the 2-aminofluorene, with or without pretreatment with butylated hydroxyanisole and butylated hydroxytoluene, DNA-2-aminofluorene adducts were observed in the target tissues of liver and bladder, and also in circulating leukocytes. The DNA-2-aminofluorene adducts in liver, bladder, and leukocytes were decreased by pretreatment with butylated hydroxyanisole and butylated hydroxytoluene. This is the first demonstration that synthetic phenolic antioxidants decrease the N-acetylation of carcinogens and formation of DNA-carcinogen adducts in vivo.

Urinary mutagenesis and fried red meat intake: influence of cooking temperature, phenotype, and genotype of metabolizing enzymes in a controlled feeding study.

Meat cooked at high temperatures contains potential carcinogenic compounds, such as heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). Samples from a 2-week controlled feeding study were used to examine the relationship between the intake of mutagenicity from meat fried at different temperatures and the levels of mutagenicity subsequently detected in urine, as well as the influence of the genotype of drug metabolizing enzymes on urinary mutagenicity. Sixty subjects consumed ground beef patties fried at low temperature (100 degrees C) for 1 week, followed by ground beef patties fried at high temperature (250 degrees C) the second week. Mutagenicity in the meat was assayed in Salmonella typhimurium TA98 (+S9), and urinary mutagenicity was determined using Salmonella YG1024 (+S9). Genotypes for NAT1, NAT2, GSTM1, and UGT1A1 were analyzed using blood samples from the subjects. Meat fried at 100 degrees C was not mutagenic, whereas meat fried at 250 degrees C was mutagenic (1023 rev/g). Unhydrolyzed and hydrolyzed urine samples were 22x and 131x more mutagenic, respectively, when subjects consumed red meat fried at 250 degrees C compared with red meat fried at 100 degrees C. We found that hydrolyzed urine was approximately 8x more mutagenic than unhydrolyzed urine, likely due to the deconjugation of mutagens from glucuronide. The intake of meat cooked at high temperature correlated with the mutagenicity of unhydrolyzed urine (r = 0.32, P = 0.01) and hydrolyzed urine (r = 0.34, P = 0.008). Mutagenicity in unhydrolyzed urine was not influenced by NAT1, NAT2, or GSTM1 genotypes. However, a UGT1A1*28 polymorphism that reduced UGT1A1 expression and conjugation modified the effect of intake of meat cooked at high temperature on mutagenicity of unhydrolyzed urine (P for interaction = 0.04). These mutagenicity data were also compared with previously determined levels of HCAs (measured as MeIQx, DiMeIQx, and PhIP) and polycyclic aromatic hydrocarbons (PAHs) in the meat, levels of HCAs in the urine, and CYP1A2 and NAT2 phenotypes. The levels of mutagenicity in the meat fried at low and high temperatures correlated with levels of HCAs, but not levels of PAHs, in the meat. Also, levels of mutagenicity in unhydrolyzed urine correlated with levels of MeIQx in unhydrolyzed urine (r = 0.36; P = 0.01), and the levels of mutagenicity of hydrolyzed urine correlated with levels of MeIQx (r = 0.34; P = 0.01) and PhIP (r = 0.43; P = 0.001) of hydrolyzed urine. Mutagenicity in unhydrolyzed urine was not influenced by either the CYP1A2 or NAT2 phenotype. The data from this study indicate that urinary mutagenicity correlates with mutagenic exposure from cooked meat and can potentially be used as a marker in etiological studies on cancer.

[Type of acetylation in children with Hodgkin's disease]. / Tip atsetilirovaniia u detei bol'nykh limfogranulematozom.

Examination of a healthy European population showed 60% to be "slow" acetylators and 40% "rapid" ones. A similar distribution was established in pediatric controls. Among children with Hodgkin's disease, there were 4.1 times more slow acetylators. N-acetyltransferase activity was found to increase with tumor advancement. Conversion of acetylation pattern was observed in pediatric patients with Hodgkin's disease.