BackgroundHeat shock proteins (Hsps) are overexpressed in several tumors and contribute to cell proliferation, metastasis, and anticancer drug resistance. Therefore, Hsp inhibitors have enhanced cytotoxicity as chemotherapeutic agents and may be effective with a reduced dosage for tumor therapy to avoid side effects.ResultsFour new azaphilones, maximazaphilones I–IV (1–4), and three known compounds (5–7) have been isolated from the airborne-derived fungus Penicillium maximae. Inhibitory effects of isolated compounds against induction of Hsp105 were evaluated by the luciferase assay system using Hsp105 promoter. In this assay, 2–4, 6, and 7 significantly inhibited hsp105 promoter activity without cytotoxicity. In addition, all isolated compounds except for 5 significantly induced the death of Adriamycin (ADR)-treated HeLa cells. Interestingly, 1–4, 6, and 7 didn’t show anti-proliferative and cell death-inducing activity without ADR.ConclusionThis study revealed the chemical structures of maximazaphilones I–IV (1–4) and the potency of azaphilones may be useful for cancer treatment and reducing the dose of anticancer agents. In addition, one of the mechanisms of cell death-inducing activity for 2–4, 6, and 7 was suggested to be inhibitory effects of Hsp105 expression.

BackgroundThe human population residing in monazite bearing Kerala coast are exposed to chronic low dose and low dose rate external gamma radiation due to Th232 deposits in its beach sand. The radiation level in this area varies from < 1.0 to 45.0 mGy/year. This area serves as an ideal source for conducting large-scale epidemiological studies for assessing risk of low dose and low dose rate radiation exposure on human population. The areas with a dose level of ≤1.50 mGy/year are considered as normal level natural radiation areas (NLNRAs) and areas with > 1.50 mGy/year, as high level natural radiation areas (HLNRAs). HLNRAs were further stratified into three dose groups of 1.51-3.0 mGy/year, 3.01-6.00 mGy/year and > 6.0 mGy/year. The present study evaluates the effects of chronic low dose radiation (LDR) exposure on the birth prevalence of Congenital Heart Diseases (CHD) among the live newborns monitored in hospital based prospective study from NLNRAs and HLNRAs of Kerala coast, India.MethodologyConsecutive newborns were monitored from two hospital units located in the study area for congenital malformations. Referred CHD cases among the newborns screened were confirmed by conducting investigations such as pulse oximetry, chest X-ray, electrocardiogram and echocardiogram etc.ResultsAmong the newborns screened, 289 CHDs were identified with a frequency of 1.49‰ among 193,634 livebirths, which constituted 6.03% of overall malformations and 16.29% of major malformations. Multiple logistic regression analysis suggested that the risk of CHD among the newborns of mothers from HLNRAs with a dose group of 1.51-3.0 mGy/year was significantly lower as compared to NLNRA (OR = 0.72, 95% CI: 0.57-0.92), whereas it was similar in HLNRA dose groups of 3.01-6.00 mGy/year (OR = 0.55, 95% CI: 0.31-1.00) and ≥ 6.0 mGy/year (OR = 0.96, 95% CI: 0.50-1.85). The frequency of CHDs did not show any radiation dose related increasing trend. However, a significant (P = 0.005) reduction was observed in the birth prevalence of CHDs among the newborns from HLNRA (1.28‰) as compared to NLNRA (1.79‰).ConclusionChronic LDR exposure did not show any increased risk on the birth prevalence of CHDs from high-level natural radiation areas of Kerala coast, India. No linear increasing trend was observed with respect to different background dose groups. The frequency of CHD was observed to be 1.49 per 1000 livebirths, which was similar to the frequency of severe CHD rate reported elsewhere in India and was much less than the reported frequency of 9 per thousand.

BackgroundThe PIG-A gene mutation assay is a valuable tool for measuring in vivo gene mutations in blood cells. The human PIG-A assay, used as a potential genotoxicity biomarker, is minimally invasive, sensitive, and cost-efficient; however, the relationship between carcinogen exposure and PIG-A mutations is not well understood.MethodsWe investigated the genotoxic effect of red blood cells using PIG-A assay and lymphocyte cytokinesis-block micronucleus test in barbecue restaurant workers (N = 70) exposed to polycyclic aromatic hydrocarbons (PAHs) and self-identified healthy control subjects (N = 56). Urinary PAH metabolites were measured to evaluate internal exposure levels.ResultsMultivariate Poisson regression showed that the PAH-exposed workers exhibited significantly higher PIG-A mutant frequency (MF) (8.04 ± 6.81 × 10− 6) than did the controls (5.56 ± 5.26 × 10− 6) (RR = 0.707, 95% CI: 0.615–0.812, P < 0.001). These results indicate that PAH exposure is a risk factor for elevated PIG-A MF. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in the PAH-exposed workers (MN: 3.06 ± 2.07 ‰, NBUD: 1.38 ± 1.02 ‰) were also significantly higher than in the controls (MN: 1.46 ± 0.64 ‰, P < 0.001; NBUD: 0.70 ± 0.60 ‰, P < 0.001). Additionally, PIG-A MFs showed better associations with several urinary hydroxylated PAH metabolites (P2-OH-Flu = 0.032, r2-OH-Flu = 0. 268; P2-OH-Phe = 0.022, r2-OH-Phe = 0.286; P3-OH-Phe = 0.0312, r3-OH-Phe = 0.270; P4-OH-Phe = 0.018, r4-OH-Phe = 0.296), while the increase in MN, NPB, and NBUD frequencies was not associated with any OH-PAH metabolites; and high-PAH-exposed workers showed the highest PIG-A MFs. Furthermore, there was a significant association between PIG-A MF and PAH exposure levels (Chi-square test for trend, P = 0.006).ConclusionsOur results indicate that an increase in PIG-A MF in barbecue workers could reflect the response to PAH exposure, providing evidence of its potential as a genotoxicity biomarker in human risk assessment.

BackgroundDNA adducts, covalent modifications to DNA due to exposure to specific carcinogens, cause the mispairing of DNA bases, which ultimately results in DNA mutations. DNA methylation in the promoter region, another type of DNA base modification, alters the DNA transcription process, and has been implicated in carcinogenesis in humans due to the down-regulation of tumor suppressor genes. Difficulties are associated with demonstrating the existence of DNA adducts or chemically modified bases in the human urological system. Apart from aristolochic acid-DNA adducts, which cause urothelial carcinoma and endemic nephropathy in a particular geographical area (Balkan), limited information is currently available on DNA adduct profiles in renal cell carcinoma and upper urinary tract urothelial carcinoma, including renal pelvic cancer and ureteral cancer.MethodTo elucidate the significance of DNA adducts in carcinogenesis in the urothelial system, we investigated 53 DNA adducts in the non-tumoral renal parenchyma and non-tumoral renal pelvis of patients with renal cell carcinoma, upper urinary tract urothelial carcinoma, and other diseases using liquid chromatography coupled with tandem mass spectrometry. A comparative analysis of tissue types, the status of malignancy, and clinical characteristics, including lifestyle factors, was performed.ResultsC5-Methyl-2′-deoxycytidine, C5-hydroxymethyl-2′-deoxycytidine (5hmdC), C5-formyl-2′-deoxycytidine, 2′-deoxyinosine, C8-oxo-2′-deoxyadenosine, and C8-oxo-2′-deoxyguanosine (8-OHdG) were detected in the renal parenchyma and renal pelvis. 8-OHdG was more frequently detected in the renal pelvis than in the renal cortex and medulla (p = 0.048 and p = 0.038, respectively). 5hmdC levels were significantly lower in the renal pelvis of urothelial carcinoma patients (n = 10) than in the urothelium of patients without urothelial carcinoma (n = 15) (p = 0.010). Regarding 5hmdC levels in the renal cortex and medulla, Spearman’s rank correlation test revealed a negative correlation between age and 5hmdC levels (r = − 0.46, p = 0.018 and r = − 0.45, p = 0.042, respectively).ConclusionsThe present results revealed a reduction of 5hmdC levels in the non-tumoral urinary tract mucosa of patients with upper urinary tract urothelial carcinoma. Therefore, the urothelial cell epithelia of patients with upper urinary tract cancer, even in non-cancerous areas, may be predisposed to urothelial cancer.

BackgroundRheumatoid arthritis (RA) is the most common autoimmune system diseases in our world. More studies in recent years have shown that FCRL gene polymorphisms is closely related to autoimmune diseases. It is suggested that genetic factors play a crucial role in the pathogenesis of this disease. In this study, we aimed to investigate the relationship between FCRL1 rs2050568, FCRL3 rs2317230 and FCRL6 rs58240276 polymorphisms and RA risk in the Chinese Han population. 506 with RA patients and 509 healthy controls were recruited in this study, and the single nucleotide polymorphisms (SNPs) was successfully genotyped using the Agena MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (95% CIs) after adjusting for age and gender were conducted to assess these SNPs polymorphisms and RA risk. The multifactor dimensionality reduction (MDR) method was conducted to analyze SNP-SNP interaction.ResultsOur results revealed that there no significant association was observed between the allele and genotype frequencies among these SNPs and RA risk (all p > 0.05). Straified analysis by age and gender, the results confirmed that FCRL1 rs2050568 T/T genotype enhanced the risk of RA in females (p = 0.014). The G/T - T/T genotype of FCRL3 rs2317230 was correlated with a decreased RA risk in males (p = 0.021). We also observed that the C/T-T/T genotype of FCRL6 rs58240276 was increased the risk of RA in the group at age > 54 years (p = 0.016). In addition, FCRL1 rs2050568-TT, FCRL6 rs58240276-TT and FCRL1 rs2050568-TT, FCRL3 rs2317230-TT, FCRL6 rs58240276-TT are the best models for multi-site MDR analysis (p < 0.05), and the two best models mentioned above and classes RA have the most significant correlation.ConclusionsOur study demonstrated that FCRL1 rs2050568, FCRL3 rs2317230, and FCRL6 rs58240276 polymorphisms were correlated with RA susceptibility in the Chinese Han population.

Chemical carcinogens to humans have been usually identified by epidemiological studies on the relationships between occupational or environmental exposure to the agents and specific cancer induction. In contrast, carcinogenic heterocyclic amines were identified under the principle that mutagens in bacterial in the Ames test are possible human carcinogens. In the 1970s to 1990s, more than 10 heterocyclic amines were isolated from pyrolysates of amino acids, proteins, meat or fish as mutagens in the Ames test, and they were demonstrated as carcinogens in rodents. In the 1980s and 1990s, we have developed derivatives of the Ames tester strains that overexpressed acetyltransferase of Salmonella typhimurium. These strains such as Salmonella typhimurium YG1024 exhibited a high sensitivity to the mutagenicity of the carcinogenic heterocyclic amines. Because of the high sensitivity, YG1024 and other YG strains were used for various purposes, e.g., identification of novel heterocyclic amines, mechanisms of metabolic activation, comparison of mutagenic potencies of various heterocyclic amines, and the co-mutagenic effects. In the 1990s and 2000s, we developed transgenic mice and rats for the detection of mutagenicity of chemicals in vivo. The transgenics were generated by the introduction of reporter genes for mutations into fertilized eggs of mice and rats. We named the transgenics as gpt delta because the gpt gene of Escherichia coli was used for detection of point mutations such as base substitutions and frameshifts and the red/gam genes of λ phage were employed to detect deletion mutations. The transgenic rodents gpt delta and other transgenics with lacI or lacZ as reporter genes have been utilized for characterization of mutagenicity of heterocyclic amines in vivo. In this review, we summarized the in vitro mutagenicity of heterocyclic amines in Salmonella typhimurium YG strains and the in vivo mutagenicity in transgenic rodents. We discussed the relationships between in vitro and in vivo mutagenicity of the heterocyclic amines and their relations to the carcinogenicity.

BackgroundThe liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds. In particular, the repeated-dose liver MN (RDLMN) assay which greatly facilitates incorporation of the liver MN assay into the general toxicity study has been developed. Usefulness of the RDLMN assay was appraised highly in the 7th International Workshops on Genotoxicity Testing (2017 in Tokyo) in that sufficient numbers and types of chemicals were studied and easy integration into the general toxicity study is preferred from the 3R’s point of view. However, it was pointed out that it is necessary to evaluate the effect of age at the start of 4-week repeated administration, since there are limited data, where only those of rats of 6 week of age at the start of administration are available.In this study, we conducted the 4-week RDLMN assay using rats of 6 and 8 weeks of age (at the start of administration) to investigate the effect of age on the liver MN inducibility. Clofibrate, a weak inducer of liver MN, was used in this study to detect the slight difference in the liver MN induction.ResultsThe liver MN induced by clofibrate was detected in both rats of 6 and 8 weeks of age at the start of administration. However, the liver MN induction was lower in rats of 8 weeks of age compared to rats of 6 weeks of age at the start of administration.ConclusionThese results suggest that the liver MN inducibility decreases with age. Therefore, we recommend the use of rats of 6 weeks of age at start of administration to reliably detect the liver MN induction in the RDLMN assay.

Since Dr. Sugimura’s discovery of heterocyclic aromatic amines (HAA) in broiled fish, many epidemiological studies have been conducted to investigate their role in human cancers, often focusing on colorectal cancer. The difficulty in measuring HAA exposure from meat and fish intake in these studies has resulted in inconsistent findings. Because studying individuals who may be particularly susceptible to the carcinogenic effects of HAA might facilitate the demonstration of a link with cancer, multiple studies have focused on individuals with the high activity phenotype for CYP1A2 and/or NAT2, the two main metabolic enzymes involved in the bioactivation of HAA. These investigations have also yielded inconsistent results. Two recent large pooled analyses of colorectal cancer studies have helped clarify the overall evidence. One was conducted in whites and reported no interaction of red meat intake and NAT2 genotype on risk in Whites. The other was conducted in Japanese and African Americans, two populations with high rates of the disease and a prevalence of the at-risk rapid NAT2 phenotype 10- and 2-fold greater than in whites, respectively. In those groups, a significant interaction was found, with the association of red meat with colorectal cancer being strongest among individuals with the rapid NAT2 phenotype, intermediate among those with the intermediate phenotype and not significant among those with the slow NAT2 phenotype. Recent research on biomarkers has focused on PhIP hair content, as a marker of exposure to HAA, and on DNA adducts using new sensitive quantitative methods, as markers of early biological effects. These advances, when brought to bear, may contribute greatly to the further elucidation of the carcinogenicity of HAA in humans.

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

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