Head-and-neck squamous cell carcinoma risk in smokers: no association detected between phenotype and AHR, CYP1A1, CYP1A2, or CYP1B1 genotype.

BACKGROUND: Head-and-neck squamous cell carcinoma (HNSCC) differs between smokers and nonsmokers in etiology and clinical presentation. Because of demonstrated unequivocal involvement in smoking-induced cancer in laboratory animals, four candidate genes--AHR, CYP1A1, CYP1A2, and CYP1B1--were selected for a clinical genotype-phenotype association study of HNSCC risk in smokers. Thirty-six single-nucleotide variants (mostly tag-SNPs) within and near these four genes [16 (AHR), 4 (CYP1A1), 4 (CYP1A2), and 12 (CYP1B1)] were chosen. METHODS: Extreme discordant phenotype (EDP) method of analysis was used to increase statistical power. HNSCC patients--having smoked 1-40 cigarette pack-years--represented the "highly-sensitive" (HS) population; heavy smokers having smoked ≥80 cigarette-pack-years without any type of cancer comprised the "highly-resistant" (HR) group. The vast majority of smokers were intermediate and discarded from consideration. Statistical tests were performed on N = 112 HS and N = 99 HR DNA samples from whole blood. CONCLUSIONS: Among the four genes and flanking regions--one haploblock, ACTTGATC in the 5' portion of CYP1B1, retained statistical significance after 100,000 permutations (P = 0.0042); among our study population, this haploblock was found in 36.4% of African-American, but only 1.49% of Caucasian, HNSCC chromosomes. Interestingly, in the 1000 Genomes Project database, frequency of this haplotype (in 1322 African and 1006 Caucasian chromosomes) is 0.356 and 0.003, respectively. This study represents an excellent example of "spurious association by population stratification". Considering the cohort size, we therefore conclude that the variant alleles chosen for these four genes, alone or in combinations, are not statistically significantly associated with risk of cigarette-smoking-induced HNSCC.

Contributions of the three CYP1 monooxygenases to pro-inflammatory and inflammation-resolution lipid mediator pathways.

All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(⁻/⁻) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B4 (LTB4) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB4 levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB4 formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.

Analysis of human CYP1A1 and CYP1A2 genes and their shared bidirectional promoter in eight world populations.

The human CYP1A1_CYP1A2 locus comprises the CYP1A1 (5,988 bp) and CYP1A2 (7,759 bp) transcribed regions, oriented head-to-head, sharing a bidirectional promoter of 23,306 bp. The older CYP1A1 gene appears more conserved and responsible for critical life function(s), whereas the younger CYP1A2 gene might have evolved more rapidly due to environmental (dietary) pressures. A population genetics study might confirm this premise. We combined 60 CYP1A1_CYP1A2 SNPs found in the present study (eight New Guinea Highlanders, eight Samoans, four Dogrib, four Teribe, four Pehuenche, and one Caucasian) with those found in a previous study (six West Africans, four Han Chinese, six Germans, four Samoans, and four Dogrib), yielding a total of 106 SNPs in 106 chromosomes. Resequencing of Oceanians plus Amerindians in the present study yielded 21 New World SNPs ( approximately 20%), of which 17 are not previously reported in any SNP database. Various tests revealed selective pressures for both genes and both haploblocks; unfortunately, differences in rates of evolution between the two genes were undetectable. Fay & Wu's H test revealed a "hitchhiking event" centered around four SNPs in the CYP1A1 3'-UTR; a study in silico identified different microRNA-binding patterns in the hitchhiked region, when the mutations were present compared with the mutations absent.

Organ-specific roles of CYP1A1 during detoxication of dietary benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental toxicants derived from sources that include cigarette smoke, petroleum distillation, gas- and diesel-engine exhaust, and charcoal-grilled food. The gastrointestinal tract is the principal route of PAH exposures, even when inhaled. The most thoroughly studied prototype of PAHs is benzo[a]pyrene (BaP), well known to be toxic, mutagenic, and carcinogenic in various tissues and cell types. This lab has previously shown that Cyp1a1(-/-) global knockout mice treated by oral administration of BaP die at 28 to 32 days with immunosuppression, whereas wild-type mice remain healthy for 1 year on high BaP doses (125 mg/kg/day). Thus, for oral BaP, CYP1A1 is more important in detoxication than in metabolic activation. After several days of oral BaP, we found surprisingly low CYP1A1 levels in liver, compared with that in small intestine; we postulated that this finding might reflect efficient detoxication of oral BaP in proximal small intestine such that significant amounts of the inducer BaP no longer reach the liver. In the present study, many parameters were therefore compared in wild-type, Cyp1a1(-/-) global knockout, intestinal epithelial cell-specific Cyp1a1 knockout, and hepatocyte-specific Cyp1a1 knockout mice as a function of long-term oral exposure to BaP. The peak of CYP1A1 (mRNA, protein) expression in liver occurred at 12 h, whereas highly induced CYP1A1 in small intestine persisted throughout the 30-day experiment. Hepatocyte-specific Cyp1a1 knockout mice remained as healthy as wild-type mice; intestinal epithelial cell-specific Cyp1a1 knockout mice behaved like Cyp1a1(-/-) mice, dying with immunosuppression approximately 30 days on oral BaP. We conclude that small intestine CYP1A1, and not liver CYP1A1, is critically important in oral BaP detoxication.

Comparing gene expression during cadmium uptake and distribution: untreated versus oral Cd-treated wild-type and ZIP14 knockout mice.

The nonessential metal cadmium (Cd) is toxic only after entering the cell. Proteins possibly relevant to intracellular Cd accumulation include the divalent metal transporter-1 (DMT1) and all 14 zinc-like iron-like protein (ZIP) importers, 10 zinc transporter (ZnT) exporters, and metallothionein chaperones MT1 and MT2. Comparing oral Cd-treated ZIP14 knockout (KO) with wild-type (WT) mice, we predicted Cd uptake and distribution would be diminished in the KO-because ZIP14 is very highly expressed in GI tract and liver; this was indeed observed for Cd content in liver. However, the reverse was found in kidney and lung from 6 or 12 h through 10 days of Cd exposure; at these times, Cd accumulation was unexpectedly greater in KO than WT mice; mRNA levels of the 27 above-mentioned genes were thus examined in proximal small intestine (PSI) versus kidney to see if these paradoxical effects could be explained by substantial alterations in any of the other 26 genes. PSI genes highly expressed in untreated WT animals included seven ZIP and five ZnT transporters, DMT1, MT1, and MT2; kidney genes included 11 ZIP and 7 ZnT transporters, DMT1, MT1, and MT2. Over 10 days of oral Cd, a bimodal response was seen for Cd content in PSI and for various mRNAs; initially, acute effects caused by the toxic metal; subsequently, the up- or down-regulation of important genes presumably to combat the sustained adversity. These data underscore the complex interplay between the gastrointestinal tract and renal proteins that might be relevant to Cd uptake and distribution in animals exposed to oral Cd.

Analysis of six SNPs of NAT2 in Ngawbe and Embera Amerindians of Panama and determination of the Embera acetylation phenotype using caffeine.

Six NAT2 single-nucleotide polymorphisms (SNPs) were analysed in 105 unrelated Ngawbe and 136 unrelated Embera Amerindians (482 chromosomes) by SNP-specific polymerase chain reaction analysis. 282C>T was the most common synonymous mutation, while 857G>A was the most frequent nonsynonymous inactivating exchange. The allelic frequency of the NAT2*5 series (containing the 341T>C exchange) was 2.4% and 9.9% for Ngawbe and Embera, respectively, five- to 20-times lower than that in Caucasians. The NAT2*6 series (590G>A) showed allelic frequencies of 0% and 3.7%, eight- to 30-times lower than in Caucasians. On the other hand, the NAT2*7 series, characterized by mutation 857G>A, had allelic frequencies (23.3% and 22.8%) that were 10-20-times higher in Amerindians than in Caucasians. Amerindians are characterized by decreased genetic diversity because they display a low number of mutated alleles (four and five for Ngawbe and Embera, respectively) that are present at low proportions (27.6% and 39%), reduced genotypic variability (seven out of 15 and 12 out of 21 possible genotypes) and low heterozygosity (40% and 55.1%) at the NAT2 locus. The NAT2 phenotype was evaluated with caffeine in a subset of 72 Embera. There were no disagreements between genotype and phenotype among rapid and slow acetylators (13/72, 18%). We conclude that, in the Embera, the analysis of three inactivating mutations was sufficient in predicting the phenotype in more than 99.5% of these subjects. NAT2 would appear to be of a selectively neutral character given that there is no evidence of adaptation to the prevailing ecology in Amerindians.

Discovery of new potentially defective alleles of human CYP2C9.

CYP2C9 is a clinically important enzyme, responsible for the metabolism of numerous clinically important therapeutic drugs. In the present study, we discovered 38 single nucleotide polymorphisms in CYP2C9 by resequencing of genomic DNA from 92 individuals from three different racial groups. Haplotype analysis predicted that there are at least 21 alleles of CYP2C9 in this group of individuals. Six new alleles were identified that contained coding changes: L19I (CYP2C9*7), R150H (CYP2C9*8), H251R (CYP2C9*9), E272G (CYP2C9*10), R335W(CYP2C9*11) and P489S (CYP2C9*12). When expressed in a bacterial cDNA expression system, several alleles exhibited altered catalytic activity. CYP2C9*11 appeared to be a putative poor metabolizer allele, exhibiting a three-fold increase in the Km and more than a two-fold decrease in the intrinsic clearance for tolbutamide. Examination of the crystal structure of human CYP2C9 reveals that R335 is located in the turn between the J and J' helices and forms a hydrogen-bonding ion pair with D341 from the J' helix. Abolishing this interaction in CYP2C9*11 individuals could destabilize the secondary structure and alter the substrate affinity. This new putative poor metabolizer (PM) allele was found in Africans. A second potentially PM allele CYP2C9*12 found in a racially unidentified sample also exhibited a modest decrease in the Vmax and the intrinsic clearance for tolbutamide in a recombinant system. Further clinical studies are needed to determine the effect of these new polymorphisms on the metabolism of CYP2C9 substrates.

ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero.

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

Search for an association between the human CYP1A2 genotype and CYP1A2 metabolic phenotype.

The genotype responsible for more than 60-fold interindividual differences in human hepatic CYP1A2 constitutive expression is not understood. Resequencing the human CYP1A1_CYP1A2 locus (39.6 kb) in five major geographically isolated subgroups recently led to the identification of 85 single nucleotide polymorphisms (SNPs), 57 of which were double-hit SNPs. Here, we attempted to correlate the CYP1A2 genotype with a metabolic phenotype. We chose 16 SNPs (all having a minor allele frequency > or =0.05 in Caucasians) to genotype 32 DNA samples (26 Caucasians, six Ethiopians) in which CYP1A2 metabolism had previously been determined. From 280 subjects (five locations worldwide) that had been CYP1A2-phenotyped, we genotyped the 10 highest, 14 lowest and eight intermediate DNA samples. Although no SNP was significant (P<0.05), possibly due to the small sample size, we found a trend for several of the six SNPs across the CYP1A2 linkage disequilibrium block associated with the trait. Five CYP1A2 haplotypes were inferred, two of which had not previously been reported; haplotype 1A2H10 showed the greatest association with CYP1A2 activity. Regulatory sequences responsible for the large interindividual differences in hepatic CYP1A2 gene basal expression might reside, in part, with some of these CYP1A2 SNPS but, in large part, might be located either cis (in nearby sequences not yet haplotyped) or trans in that they are not linked to the gene. We conclude that no SNP or haplotype in the CYP1A2 gene has yet been identified that can unequivocally be used to predict the metabolic phenotype in any individual patient.