Genetic Risk Factors Associated With Antiemetic Efficacy of Palonosetron, Aprepitant, and Dexamethasone in Japanese Breast Cancer Patients Treated With Anthracycline-based Chemotherapy.

INTRODUCTION: Breast cancer patients often receive anthracycline-based chemotherapy, and chemotherapy-induced nausea and vomiting (CINV) remains one of the most uncomfortable and distressing adverse reactions. Poor control of CINV reduces the relative dose intensity of chemotherapy agents, which has been associated with poor clinical outcomes and shorter survival. The aim of the present study was to identify genetic risk factors associated with anthracycline-based CINV. PATIENTS AND METHODS: We evaluated CINV attributable to anthracycline-based chemotherapy in Japanese breast cancer patients treated with an antiemetic regimen that included palonosetron, aprepitant, and dexamethasone. Furthermore, we investigated the associations between CINV and single nucleotide polymorphisms in 6 candidate genes. RESULTS: Emesis episodes were rarely observed in the 125 patients included in the present survey (7.2%; n = 9); however, significant nausea occurred in more than one half of the patients (52.8%; n = 66). In particular, acute significant nausea was not effectively controlled. Multivariate logistic regression analysis revealed that the ABCG2 (rs2231142) AA genotype is significantly associated with acute significant nausea (odds ratio, 4.87; 95% confidence interval, 1.01-23.60; P = .049). CONCLUSION: The findings of the present study provide significant insights for developing personalized antiemetic strategies for breast cancer patients receiving anthracycline-based chemotherapy.

Dioxin-metabolizing genes in relation to effects of prenatal dioxin levels and reduced birth size: The Hokkaido study.

OBJECTIVES: We investigated the effects of maternal polymorphisms in 3 genes encoding dioxin-metabolizing enzymes in relation to prenatal dioxin levels on infant birth size in Japan. METHODS: We examined the relationship between dioxin exposure and birth size in relation to the polymorphisms in the genes encoding aromatic hydrocarbon receptor (AHR [G>A, Arg554Lys]), cytochrome P450 (CYP) 1A1 (T6235C), and glutathione S-transferase mu 1 (GSTM1; Non-null/null) in 421 participants using multiple linear regression models. RESULTS: In mothers carrying the GSTM1 null genotype, a ten-fold increase in total dioxin toxic equivalency was correlated with a decrease in birth weight of -345g (95% confidence interval: -584, -105). CONCLUSIONS: We observed adverse effects of maternal GSTM1 null genotype on birth weight in the presence of dioxins exposure during pregnancy.

Genetic association of aromatic hydrocarbon receptor (AHR) and cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) polymorphisms with dioxin blood concentrations among pregnant Japanese women.

Dioxins are metabolized by cytochrome P450, family 1 (CYP1) via the aromatic hydrocarbon receptor (AHR). We determined whether different blood dioxin concentrations are associated with polymorphisms in AHR (dbSNP ID: rs2066853), AHR repressor (AHRR; rs2292596), CYP1 subfamily A polypeptide 1 (CYP1A1; rs4646903 and rs1048963), CYP1 subfamily A polypeptide 2 (CYP1A2; rs762551), and CYP1 subfamily B polypeptide 1 (CYP1B1; rs1056836) in pregnant Japanese women. These six polymorphisms were detected in 421 healthy pregnant Japanese women. Differences in dioxin exposure concentrations in maternal blood among the genotypes were investigated. Comparisons among the GG, GA, and AA genotypes of AHR showed a significant difference (genotype model: P=0.016 for the mono-ortho polychlorinated biphenyl concentrations and toxicity equivalence quantities [TEQs]). Second, we found a significant association with the dominant genotype model ([TT+TC] vs. CC: P=0.048 for the polychlorinated dibenzo-p-dioxin TEQs; P=0.035 for polychlorinated dibenzofuran TEQs) of CYP1A1 (rs4646903). No significant differences were found among blood dioxin concentrations and polymorphisms in AHRR, CYP1A1 (rs1048963), CYP1A2, and CYP1B1. Thus, polymorphisms in AHR and CYP1A1 (rs4646903) were associated with maternal dioxin concentrations. However, differences in blood dioxin concentrations were relatively low.

Genetic polymorphisms of 17 ß-hydroxysteroid dehydrogenase 3 and the risk of hypospadias.

INTRODUCTION: Hypospadias is a common congenital anomaly caused by incomplete fusion of urethral folds. Development of the urethra and external genital system in the male fetus is an androgen-dependent process. In this regard, enzymes 17 ß-hydroxysteroid dehydrogenase type 3 (17 ß HSD3, encoded by HSD17B3) and steroid 5 α-reductase type 2 (encoded by SRD5A2) play crucial roles. AIM: To investigate the possible associations between common polymorphisms in HSD17B3 as well as well-known V89L polymorphism in SRD5A2 and risk of hypospadias. METHODS: A case-control study was performed between 1999 and 2005. There were 89 Japanese boys with hypospadias and 291 newborn controls. We genotyped HSD17B3-1999T>C, +10A>G, +20A>G, +139G>A (V31I), +913G>A (G289S), and SRD5A2+336G>C (V89L) polymorphisms by allelic discrimination assay. We measured mRNA expression of the wildtype G289 allele and the mutant S289 allele of the HSD17B3 gene in the transfected human fetal kidney HEK293 cells. MAIN OUTCOME MEASURES: Assessment of hypospadias including its severity and HSD17B3 and SRD5A2 genes using DNA blood samples: allele and genotype distribution of single nucleotide polymorphisms in these two genes in cases and controls. RESULTS: In our study, the risk of hypospadias was significantly higher in subjects carrying homozygous HSD17B3+913A (289S) alleles (odds ratio [OR]: 3.06; 95% confidence interval [CI]: 1.38-6.76). The risk of severe hypospadias was much higher in these subjects (OR: 3.93; 95% CI: 1.34-11.49). The mRNA expression levels of HSD17B3 G289 were higher than those of HSD17B3 S289 mutant (P < 0.001). In addition, the risk of severe hypospadias increased in boys carrying the SRD5A2+336C (89L) allele (OR: 3.19; 95% CI: 1.09-9.36). CONCLUSIONS: These results suggest that the HSD17B3 G289S polymorphism may be a potential risk modifier for hypospadias. Our findings provide evidence that a certain genotype related to androgen production may potentiate risk of hypospadias.

Prenatal exposure to PCDDs/PCDFs and dioxin-like PCBs in relation to birth weight.

Several human studies have shown that low-level exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs) and organochlorine pesticides, negatively influences birth outcomes. However, the effects of low-level exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like PCBs (DL-PCBs) on birth outcomes have not been clarified in human studies. A prospective cohort study was established to investigate the possible adverse effects of PCDDs/PCDFs and DL-PCBs on fetal growth and neurodevelopment. We recruited 514 pregnant women between July 2002 and October 2005 in Sapporo, Japan. We measured 29 congener levels of PCDDs/PCDFs and DL-PCBs in maternal blood. Using multiple liner regression analysis of the association between birth weight and the levels of PCDDs/PCDFs and DL-PCBs with full adjustments for potential confounders, a significant adverse effect was observed regarding total PCDDs toxic equivalents (TEQ) levels (adjusted beta=-231.5g, 95% CI: -417.4 to -45.6) and total PCDFs TEQ levels (adjusted beta=-258.8g, 95% CI: -445.7 to -71.8). Among male infants, significant adverse associations with birth weight were found for total PCDDs TEQ level, total PCDDs/PCDFs TEQ level, and total TEQ level. However, among female infants, these significant adverse associations were not found. With regard to individual congeners of PCDDs/PCDFs and DL-PCBs, we found significantly negative association with the levels of 2,3,4,7,8-PeCDF (adjusted beta=-24.5g, 95% CI: -387.4 to -61.5). Our findings suggest that prenatal low-level exposure to PCDDs and PCDFs, especially 2,3,4,7,8-PeCDF, may accumulate in the placenta and retard important placental functions, which result in lower birth weight.

Correlations between prenatal exposure to perfluorinated chemicals and reduced fetal growth.

BACKGROUND: Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are man-made, ubiquitous, and persistent contaminants in the environment, wildlife, and humans. Although recent studies have shown that these chemicals interfere with fetal growth in humans, the results are inconsistent. OBJECTIVES: Our goal was to investigate the correlation between relatively low levels of PFOS and PFOA in maternal serum and birth weight and birth size. METHODS: We conducted a hospital-based prospective cohort study between July 2002 and October 2005 in Sapporo, Japan. A total of 428 women and their infants were involved in the study. We obtained characteristics of the mothers and infants from self-administered questionnaire surveys and from medical records. We analyzed maternal serum samples for PFOS and PFOA by liquid chromatography-tandem mass spectrometry (LC/MS/MS). RESULTS: After adjusting for confounding factors, PFOS levels negatively correlated with birth weight [per log10 unit: beta = -148.8 g; 95% confidence interval (CI), -297.0 to -0.5 g]. In addition, analyses stratified by sex revealed that PFOS levels negatively correlated with birth weight only in female infants (per log10 unit: beta = -269.4 g; 95% CI, -465.7 to -73.0 g). However, we observed no correlation between PFOA levels and birth weight. CONCLUSION: Our results indicate that in utero exposure to relatively low levels of PFOS was negatively correlated with birth weight.

Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

Genetic polymorphisms of ESR1 and ESR2 that may influence estrogen activity and the risk of hypospadias.

BACKGROUND: The etiology of hypospadias is regarded as a complex disorder with both genetic and environmental contributions. Although alterations in androgen activity have been associated with hypospadias, few associations with estrogen activity have been documented. Here, we assessed genetic polymorphisms in estrogen receptor genes and their association with hypospadias. METHODS: Using a case-control study of 59 cases with hypospadias and 286 controls, we examined the association of hypospadias with the following polymorphisms: PvuII and XbaI in ESR1, and 2681-4A>G in ESR2. RESULTS: For the cases, we found a negative association with the G allele containing variants of ESR1 XbaI (OR = 0.52, P < 0.05), and a negative association with the G allele containing variants of ESR2 2681-4A>G (OR = 0.59, P < 0.05). For the cases, we also identified a negative association with the CG haplotype, and a positive association with the CA haplotype, defined by ESR1 PvuII and XbaI (P < 0.05). CONCLUSIONS: These findings suggest that the G allele containing variants of ESR1 XbaI and the G allele containing variants of ESR2 2681-4A>G may decrease the risk of hypospadias, whereas the ESR1 C-A haplotype may increase its risk.

Adverse birth outcomes associated with maternal smoking and polymorphisms in the N-Nitrosamine-metabolizing enzyme genes NQO1 and CYP2E1.

Maternal smoking during pregnancy can result in both pregnancy complications and reduced size of the fetus and neonate. Among women who smoke, genetic susceptibility to tobacco smoke also is a likely causative factor in adverse pregnancy outcomes. A prospective cohort study was conducted among 460 pregnant women who delivered live singletons in Sapporo, Japan, from 2002 to 2005. Multiple linear regression models were used to estimate associations of maternal smoking and polymorphisms in two genes encoding N-nitrosamine-metabolizing enzymes-NAD(P)H: quinone oxidoreductase 1 (NQO1) and cytochrome P-450 2E1 (CYP2E1)-with birth size. Among infants born to smokers with the NQO1 homozygous wild-type allele, birth weight, birth length, and birth head circumference were significantly reduced (p < 0.01 for each factor). For the homozygous wild-type CYP2E1 allele, birth weight was lower by an estimated 195 g (standard error, 55; p < 0.001) among smokers. These genotypes did not confer adverse effects among women who had never smoked or who quit smoking during the first trimester. The adverse effects of maternal smoking on infant birth size may be modified by maternal genetic polymorphisms in N-nitrosamine-metabolizing enzymes among Japanese subjects. These results may help in directing smoking cessation interventions during pregnancy, especially among susceptible women.

Exploiting gene-environment interaction to detect adverse health effects of environmental chemicals on the next generation.

There is increasing evidence from epidemiological studies that genetic susceptibilities may modify the teratogenic effects of toxic chemicals. However, in contrast to tobacco smoke, few epidemiological studies have addressed environmental chemicals, such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls in regard to genetic susceptibility. Recent studies, including the Hokkaido Study of Environments and Children's Health, have investigated the impacts of both environmental and genetic factors on children's development. Several xenobiotic-metabolizing genes have been reported to confer genetic susceptibility to low birth weight. These genes seem to be influenced functionally by maternal smoking during pregnancy, itself a significant risk factor. In our study, we found that birth weight was significantly lower among infants born to smoking women having the specific AHR, CYP1A1, GSTM1, CYP2E1 and NQO1 genotypes. When combinations of these genotypes were considered, birth weight was even lower. On the other hand, congenital anomalies such as hypospadias seemed to be caused by environmental factors in conjunction with genetic predisposition as suggested by linkage in several case-control studies reported to low birth weight. We have found an association between maternal CYP1A1 genotype or low birth weight and the risk of hypospadias irrespective of smoking. At the same time, birth weight was negatively correlated with maternal blood concentrations of polychlorinated dibenzofurans. Further studies should elucidate the impact of genetic factors on adverse effects of exposures to dioxin-related chemicals.

Using microarray analysis to evaluate genetic polymorphisms involved in the metabolism of environmental chemicals.

The field of molecular biology currently faces the need for a comprehensive method of evaluating individual differences derived from genetic variation in the form of single nucleotide polymorphisms (SNPs). SNPs in human genes are generally considered to be very useful in determining inherited genetic disorders, susceptibility to certain diseases, and cancer predisposition. Quick and accurate discrimination of SNPs is the key characteristic of technology used in DNA diagnostics. For this study, we first developed a DNA microarray and then evaluated its efficacy by determining the detection ability and validity of this method. Using DNA obtained from 380 pregnant Japanese women, we examined 13 polymorphisms of 9 genes, which are associated with the metabolism of environmental chemical compounds found in high frequency among Japanese populations. The ability to detect CYP1A1 I462V, CYP1B1 L432V, GSTP1 I105V and AhR R554K gene polymorphisms was above 98%, and agreement rates when compared with real time PCR analysis methods (kappa values) showed high validity: 0.98 (0.96), 0.97 (0.93), 0.90 (0.81), 0.90 (0.91), respectively. While this DNA microarray analysis should prove important as a method for initial screening, it is still necessary that we find better methods for improving the detection of other gene polymorphisms not part of this study.

Exposure of prepubertal female rats to inhaled di(2-ethylhexyl)phthalate affects the onset of puberty and postpubertal reproductive functions.

We evaluated the effects of inhaled di(2-ethylhexyl)phthalate (DEHP) on the onset of puberty and on postpubertal reproductive functions in prepubertal female rats. DEHP was administered by inhalation at doses of 0, 5, and 25 mg/m3 to groups of female rats for 6 h/day, 5 contiguous days/week from postnatal days (PNDs) 22 to 41 and to PND 84. The onset of puberty was determined by daily examination for vaginal opening (VO) and first estrous cycle. Reproductive function was evaluated by observing estrous cyclicity from PNDs 49 to 84. Upon completion of exposure, the rats were sacrificed at PND 42 and PNDs 85-88 during the diestrous stage. DEHP exposure advanced the age of VO and first estrous cycle, and serum cholesterol, luteinizing hormone, and estradiol levels were significantly elevated in the 25-mg/m3 DEHP group. Irregular estrous cycles were observed more frequently in DEHP-exposed rats, and serum cholesterol decreased in DEHP-exposed rats in adulthood; RT-PCR showed that the expression of aromatase mRNA, encoding a rate-limiting enzyme that catalyzes the conversion of testosterone to estradiol, was elevated in the 25-mg/m3 DEHP group. These data suggest that inhaled DEHP may advance the onset of puberty and alter postpubertal reproductive functions.

Highly polymorphic vitelline-coat protein HaVC80 from the ascidian, Halocynthia aurantium: structural analysis and involvement in self/nonself recognition during fertilization.

Ascidians release sperm and eggs simultaneously, but self-fertilization is effectively blocked by unknown mechanisms. We previously reported that a 70-kDa sperm receptor HrVC70 on the egg vitelline coat (VC) consisting of 12 EGF-like repeats is a candidate self/nonself recognition molecule during fertilization of the ascidian, Halocynthia roretzi. Here, we report that Halocynthia aurantium also utilizes a homolog (HaVC80) of HrVC70 as an allorecognizable sperm receptor. HaVC80 is attached to the VC during the acquisition of self-sterility and is detached from the VC by acid treatment, allowing self-fertilization. A cDNA clone of the HaVC80 precursor, HaVC130, consists of 3726 nucleotides and encodes an open reading frame of 1208 amino acids. The structure of HaVC130 is very similar to the HrVC70 precursor HrVC120, but the number of EGF-like repeats of HaVC130/VC80 is one repeat larger than that of HrVC120/VC70. There are several amino acid substitutions between different individuals, and two alleles of the HaVC80 sequence were detected in each individual. Genomic DNA sequence analysis reveals that each EGF-like domain corresponds to a specific exon, and HaVC130 may have been evolutionarily generated from HrVC120 by duplication of the 8th EGF-like repeat. The data support the hypothesis that HaVC80 is a highly polymorphic protein responsible for self-sterility in H. aurantium.

Self/nonself recognition in ascidian fertilization: vitelline coat protein HrVC70 is a candidate allorecognition molecule.

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self/nonself-recognition system in spermegg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self/nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even- and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.

Expression of trypsin-like proteases and protease nexin-1 in mdx mouse muscles.

In order to examine the possible participation of trypsin-like proteases in the onset and progress of muscular dystrophy, we investigated the expression of the trypsin-like protease in muscular tissues in mdx mice. We found that the mRNAs of several trypsin-like proteases, including hepsin and t-PA, were expressed in the muscular tissues of mdx mice, but at levels not significantly different from normal mice. Since the enzymatic properties of dystrypsin, a muscle trypsin-like protease activated before onset of the disease, are similar to those of thrombin, we investigated the expression pattern of thrombin in mdx mouse muscles. The results showed that prothrombin mRNA is up-regulated in mdx mice at 20-30 days of age but not before the age of 15 days (preclinical). Since protease nexin-1 (PN-1) is known to be a physiological inhibitor of thrombin, we also examined the expression pattern of PN-1. We found that PN-1 transcription and translation is down-regulated in the muscular tissues of mdx mice, before the onset of clinical symptoms. These results suggest that thrombin may be involved in the progression of muscular dystrophy or the regeneration of muscle fibers after the onset of the disease and that the reduced level of PN-1 may enhance the activities stimulate the activities of muscle proteases, including dystrypsin, at a preclinical stage in mdx mice.

Therapeutic effect of camostat mesilate on Duchenne muscular dystrophy in mdx mice.

Duchenne muscular dystrophy is known to be caused by a defective gene of dystrophin, a 427-kDa cytoskeletal protein, but the effective therapeutic drug is presently unavailable. We previously reported that a trypsin-like protease designated as dystrypsin is markedly activated in the muscle microsomal fraction immediately before onset of the clinical signs in mdx mice, a dystrophin-deficient hereditary animal model for human Duchenne muscular dystrophy. In order to examine the possible participation of dystrypsin in the occurrence of the disease, we investigated the therapeutic effects of dystrypsin inhibitors on the occurrence and progress of muscular dystrophy. Here, we show that camostat mesilate, a low-molecular-weight inhibitor of trypsin-like proteases, including dystrypsin, is a candidate drug for Duchenne muscular dystrophy.