Potent Environmental Carcinogen Research Articles

Abstract 6458: Maternal and paternal alcohol exposures program higher incidence of hepatocellular carcinoma in offspring

Abstract Environmental exposures during development are known to influence adult health and disease. Specifically, early developmental exposures have been shown to increase susceptibility to cancer in adults through epigenetic mechanisms. Alcohol is a potent environmental carcinogen and teratogen, causing a wide range of health problems across the life course of an individual. Current literature examining early life exposures solely focuses on maternal exposures despite recent efforts to recognize paternal epigenetic contributions. Thus, we investigated the influence of preconception paternal, maternal, and dual parental alcohol exposures on offspring’s predisposition to develop liver disease and hepatocellular carcinoma (HCC). We hypothesized that dual-parental alcohol exposure would exacerbate the incidence of liver cancer in adult offspring. Using an established mouse model, we exposed male and female adult C57BL/6J mice to control (0% EtOH) or alcohol (10% EtOH) preconception treatments. Next, we randomly assigned pups from each treatment to either a single injection of Diethylnitrosamine (DEN; 25mg/kg) to promote liver tumors, or saline control. Finally, we sacrificed offspring on postnatal day 182 and collected organs and blood for enzyme analysis. DEN-treated males display an increased liver-to-body weight ratio compared to saline males while also exhibiting lower body weight. The male progeny from the dual-parental treatment group exhibited a significant increase in tumor incidence and burden in DEN-treated males, exceeding those measured for the maternal and paternal treatments. Further, offspring from the dual-parental group had high levels of alanine transaminase (ALT) and aspartate transaminase (AST) compared to the other treatment groups. Oil Red-O and Sirius Red staining of liver sections revealed increased hepatic steatosis and fibrosis in DEN males compared to saline males. Again, the dual-parental exposed offspring exhibited the most aberrant phenotype. Blind scoring of H&E-stained liver sections identified increases in steatosis, fibrosis, and HCC lesions, with the highest scores emerging in DEN-treated dual-parental offspring. These findings demonstrate that preconception paternal and gestational maternal alcohol exposures program a predisposition to adult-onset chronic diseases including alcoholic fatty liver disease, hepatic fibrosis, and HCC. These experiments not only confirm our hypothesis that dual-parental exposed offspring exhibit an exacerbated hepatic abnormality phenotype, but also highlight the importance of both maternal and paternal environmental exposures on disease risk in offspring. While the negative effects of maternal alcohol consumption have been well established, our novel finding that paternal alcohol exposure also promotes hepatocellular carcinoma in offspring has potentially profound clinical implications. Citation Format: Alison Basel, Sanat S. Bhadsavle, Kara N. Thomas, Katherine N. Zimmel, Alexis N. Roach, Matthew Gaytan, Grace Parkey, Michael C. Golding. Maternal and paternal alcohol exposures program higher incidence of hepatocellular carcinoma in offspring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6458.

Squamous Cancer of the Esophagus in Africa: A Causal Pathway Established

Squamous cancer of the esophagus has been endemic in much of East, Central and Southern Africa since the 1940s. Much research has concentrated on attempts to identify major carcinogenic influences, and failure to do so has made it clear that the problem in high incidence areas (HIAs) is not primarily of potent environmental carcinogens, but of population susceptibility. In Africa the association with maize is constant and strong. Research in the last decade has helped to explain that association. Considered along with historical findings there is now enough evidence to establish causal associations of a nutritionally deficient maize diet and use of maize meal with squamous cancer of the esophagus. Evidence is available in a high incidence area of degenerating maize meal resulting in excess production of PGE2, gastric hypochlorhydria and a predominant pattern of non-acid gastroesophageal reflux. This pathway explains the existence of major population susceptibility: a poor maize-based diet provides specific nutritional deficiencies and n-6 fatty acid dominance which cause failure of homeostasis of the arachidonic acid cascade; with this background chemically degenerating maize meal then triggers duodenogastric reflux and non-acid gastro-esophageal reflux; non-acid reflux causes squamous cancer of the esophagus. Within a susceptible population, environmental carcinogens including tobacco increase individual risk. There is sufficient evidence that this is an active pathway, and the dominant pathway to SCCE in high incidence areas in Africa.There is sufficient evidence to justify appropriate preventative measures.

Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43mM ethanol and 25nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.

Genotoxic C8-Arylamino-2'-deoxyadenosines Act as Latent Alkylating Agents to Induce DNA Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) are extremely deleterious and structurally diverse, driving the evolution of ICL repair pathways. Discovering ICL-inducing agents is, thus, crucial for the characterization of ICL repair pathways and Fanconi anemia, a genetic disease caused by mutations in ICL repair genes. Although several studies point to oxidative stress as a cause of ICLs, oxidative stress-induced cross-linking events remain poorly characterized. Also, polycyclic aromatic amines, potent environmental carcinogens, have been implicated in producing ICLs, but their identities and sequences are unknown. To close this knowledge gap, we tested whether ICLs arise by the oxidation of 8-arylamino-2'-deoxyadenosine (ArNHdA) lesions, adducts produced by arylamino carcinogens. Herein, we report that ArNHdA acts as a latent cross-linking agent to generate ICLs under oxidative conditions. The formation of an ICL from 8-aminoadenine, but not from 8-aminoguanine, highlights the specificity of 8-aminopurine-mediated ICL production. Under the influence of the reactive oxygen species (ROS) nitrosoperoxycarbonate, ArNHdA (Ar = biphenyl, fluorenyl) lesions were selectively oxidized to generate ICLs. The cross-linking reaction may occur between the C2-ArNHdA and N2-dG, presumably via oxidation of ArNHdA into a reactive diiminoadenine intermediate followed by the nucleophilic attack of the N2-dG on the diiminoadenine. Overall, ArNHdA-mediated ICLs represent rare examples of ROS-induced ICLs and polycyclic aromatic amine-mediated ICLs. These results reveal novel cross-linking chemistry and the genotoxic effects of arylamino carcinogens and support the hypothesis that C8-modified adenines with low redox potential can cause ICLs in oxidative stress.

Benzo[a]pyrene represses DNA repair through altered E2F1/E2F4 function marking an early event in DNA damage-induced cellular senescence.

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.

The HONO-methamphetamine adduct – An unexpected derivative

The public health risks associated with methamphetamine contamination in residential homes is of concern in many jurisdictions. Current regulatory remediation guidelines target the removal of methamphetamine as it is the primary residue found at sites of illicit manufacture and/or use of the drug. However, other substances present, including decomposition products of methamphetamine, may also be of health concern. Identification of such decomposition products will allow scientists to gain a deeper understanding of the degradation mechanisms of methamphetamine in order to identify compounds which may pose risks to public health and to determine more efficient decontamination methods. Controlled oxidative reaction of methamphetamine residue by nitrous acid (HONO) gas, a ubiquitous indoor pollutant, caused the potent environmental carcinogen and potential human mutagen N-nitrosomethamphetamine (NMA) to form. In order to develop a method that could detect this compound in field samples from suspected sites of methamphetamine contamination, samples of NMA were deposited onto wiping media and processed following typical extraction and derivatisation protocols used for methamphetamine surface wipe analysis. NMA was observed to undergo chemical derivatisation with the fluorinated derivatising agent, trifluoroacetic anhydride, to form 2,2,2-trifluoro-N′-formyl-N′-(1-phenylpropan-2-yl)acetohydrazide. The structure was confirmed by synthesis of the derivative and characterisation using GC–MS, LC–MS, and NMR. Detection of NMA at sites with methamphetamine contamination would imply that there is a necessity for additional research to be conducted to ensure that public health is sufficiently protected.

Integrated extraction approach for trace analysis of aflatoxin B1 in domestic water tanks using HPLC

AbstractAnalytical strategies need to evolve to maintain high level of vigilance for domestic water toxins. A key aspect could be the integrated extraction approaches with the best combination of two extraction techniques which can quantify trace levels of toxins in water samples. Herein, we demonstrate a simple C18 based integrated solid phase extraction and micro‐extraction by packed sorbent extraction protocol coupled with high performance liquid chromatography for trace level detection and quantification of aflatoxin B1 in domestic water tanks. Aflatoxin B1 is a naturally occurring mycotoxin produced by Aspergillus Fungi and is a potent environmental carcinogen. The integrated extraction strategy succeeded to push the quantification limits up to pg/mL in spiked samples. This is the first demonstration of such type of analytical integration for the trace analysis in water. The optimized analytical protocol with a linear range of 0.05–50 ng/mL shows a detection limit of 0.012 ng/mL and a quantification limit of 0.039 ng/mL, with recovery range of 98.01–98.65%. With real sample applications, aflatoxin B1 was quantified in the range of 3.993–38.785 ng/mL in domestic water tanks.

Elevated arsenic exposure and efficient arsenic metabolism in indigenous women around Lake Poopó, Bolivia

Elevated concentrations of inorganic arsenic, one of the most potent environmental toxicants and carcinogens, have been detected in well water around Lake Poopó, Bolivia. This study aimed to assess human exposure to arsenic in villages around Lake Poopó, and also to elucidate whether the metabolism and detoxification of arsenic in this population is as efficient as previously indicated in other Andean areas. We recruited 201 women from 10 villages around Lake Poopó. Arsenic exposure was determined as the sum concentration of arsenic metabolites (inorganic arsenic; monomethylarsonic acid, MMA; and dimethylarsinic acid, DMA) in urine (U-As), measured by HPLC-HG-ICP-MS. Efficiency of arsenic metabolism was assessed by the relative fractions of the urinary metabolites. The women had a wide variation in U-As (range 12–407 μg/L, median 65 μg/L) and a markedly efficient metabolism of arsenic with low %MMA (median 7.7%, range: 2.2–18%) and high %DMA (80%, range: 54–91%) in urine. In multivariable-adjusted linear regression models, ethnicity (Aymara-Quechua vs. Uru), body weight, fish consumption and tobacco smoking were associated with urinary arsenic metabolite fractions. On average, the Uru women had 2.5 lower % (percentage unit) iAs, 2.2 lower %MMA and 4.7 higher %DMA compared with the Aymara-Quechua women. Our study identified several factors that may predict these women's arsenic methylation capacity, particularly ethnicity. Further studies should focus on mechanisms underlying these differences in arsenic metabolism efficiency, and its importance for the risk of arsenic-related health effects.

Erratum to: S-(3-Aminobenzanthron-2-yl)cysteine in the globin of rats as a novel type of adduct and possible biomarker of exposure to 3-nitrobenzanthrone, a potent environmental carcinogen.

3-Nitrobenzanthrone (3-NBA), a potent environmental mutagen and carcinogen, is known to be activated in vivo to 3-benzanthronylnitrenium ion which forms both NH and C2-bound adducts with DNA and also reacts with glutathione giving rise to urinary 3-aminobenzanthron-2-ylmercapturic acid. In this study, acid hydrolysate of globin from rats dosed intraperitoneally with 3-NBA was analysed by HPLC/MS to identify a novel type of cysteine adduct, 3-aminobenzanthron-2-ylcysteine (3-ABA-Cys), confirmed using a synthesised standard. The 3-ABA-Cys levels in globin peaked after single 3-NBA doses of 1 and 2 mg/kg on day 2 to attain 0.25 and 0.49 nmol/g globin, respectively, thereafter declining slowly to 70–80% of their maximum values during 15 days. After dosing rats for three consecutive days with 1 mg 3-NBA/kg a significant cumulation of 3-ABA-Cys in globin was observed. 3-ABA-Cys was also found in the plasma hydrolysate. Herein, after dosing with 1 and 2 mg 3-NBA/kg the adduct levels peaked on day 1 at 0.15 and 0.51 nmol/ml plasma, respectively, thereafter declining rapidly to undetectable levels on day 15. In addition, sulphinamide adducts were also found in the exposed rats, measured indirectly as 3-aminobenzanthrone (3-ABA) split off from globin by mild acid hydrolysis. Levels of both types of adducts in the globin samples parallelled very well with 3-ABA/3-ABA-Cys ratio being around 1:8. In conclusion, 3-ABA-Cys is the first example of arylnitrenium-cysteine adduct in globin representing a new promising class of biomarkers to assess cumulative exposures to aromatic amines, nitroaromatics and heteroaromatic amines.

S-(3-Aminobenzanthron-2-yl)cysteine in the globin of rats as a novel type of adduct and possible biomarker of exposure to 3-nitrobenzanthrone, a potent environmental carcinogen.

3-Nitrobenzanthrone (3-NBA), a potent environmental mutagen and carcinogen, is known to be activated in vivo to 3-benzanthronylnitrenium ion which forms both NH and C2-bound adducts with DNA and also reacts with glutathione giving rise to urinary 3-aminobenzanthron-2-ylmercapturic acid. In this study, acid hydrolysate of globin from rats dosed intraperitoneally with 3-NBA was analysed by HPLC/MS to identify a novel type of cysteine adduct, 3-aminobenzanthron-2-ylcysteine (3-ABA-Cys), confirmed using a synthesised standard. The 3-ABA-Cys levels in globin peaked after single 3-NBA doses of 1 and 2mg/kg on day 2 to attain 0.25 and 0.49nmol/g globin, respectively, thereafter declining slowly to 70-80% of their maximum values during 15days. After dosing rats for three consecutive days with 1mg 3-NBA/kg a significant cumulation of 3-ABA-Cys in globin was observed. 3-ABA-Cys was also found in the plasma hydrolysate. Herein, after dosing with 1 and 2mg 3-NBA/kg the adduct levels peaked on day 1 at 0.15 and 0.51nmol/ml plasma, respectively, thereafter declining rapidly to undetectable levels on day 15. In addition, sulphinamide adducts were also found in the exposed rats, measured indirectly as 3-aminobenzanthrone (3-ABA) split off from globin by mild acid hydrolysis. Levels of both types of adducts in the globin samples parallelled very well with 3-ABA/3-ABA-Cys ratio being around 1:8. In conclusion, 3-ABA-Cys is the first example of arylnitrenium-cysteine adduct in globin representing a new promising class of biomarkers to assess cumulative exposures to aromatic amines, nitroaromatics and heteroaromatic amines.

Occurrence of Aflatoxins in Imported Rice at Supermarkets of Ahvaz City, Khuzestan Province, Iran

Background: Rice is one of the major cereals that are primarily consumed by humans. It may become contaminated with aflatoxins (AFs). Aflatoxin B 1 (AFB 1 ) is known as one of the most potent environmental mutagens and carcinogens. Objectives: This study was carried out to determine the concentration of AFs in the rice currently sold at the supermarkets in the city of Ahvaz, Khuzestan Province, Iran. Materials and Methods: The levels of AFs in 90 collected imported rice samples after clean-up by AflaTest columns were measured by using a High-Performance Liquid Chromatographer equipped with a C18 column, fluorescence detector (excitation 360 nm and emission 440 nm) and post-column bromide derivatization method, mobile phase of water-acetonitrile-methanol (600:200:200 v/v) +119 mg potassium bromide +100 µL con. HNO 3 at a flow rate of 1 mL/min. Results: The results showed that the highest concentration of AFB 1 and total AFs in the rice samples were 2.3500 and 2.7040 ng/g, respectively. The different mean concentration of AFB 1 and total AFs in three brands of the rice samples was significantly lower than the maximum tolerable level (MTL) of AFB 1 (5 ng/g) and total AFs (30 ng/g) set by the Institute of Standards and Industrial Research of Iran. Conclusions: In all of the investigated imported rice samples, the level of AFB 1 and total AFS were found to be lower than the Iranian MTL, and no health risk for consumers were detected at these levels of contamination.

Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid

Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

Dibenzo[def,p]Chrysene (DBC) Suppresses Antibody Formation in Spleen Cells Following Oral Exposures of Mice

Dibenzo[def,p]chrysene (DBC) is a potent environmental carcinogen in rodents, fish, and human cells examined in culture. There are numerous similarities between the patterns of cytochrome P-450 (P450) activation of DBC and its covalent binding to DNA and proteins with another polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA). Our lab has previously shown that DMBA produces immunosuppression in rodents and human cell systems. Therefore, the purpose of these studies was to examine the immunotoxicity of DBC in a rodent model that was found to be sensitive to the immunosuppressive effects of DMBA. Data showed that DBC had similar potency to DMBA in producing suppression of a T-dependent antibody response (TDAR) and altered spleen cell subsets in a similar manner as DMBA when DMBA was given by gavage for 5 d in corn oil to mice at doses of 1–100 mg/kg total cumulative doses. T-cell-independent antigen (TNP-Ficoll) responses were quantitatively less sensitive to DBC suppression. It was also found that as with DMBA, DBC produced a persistent immunosuppression, which lasted for at least 4 wk following dosing with a novel pill method for self-administration of DBC. In conclusion, DBC appears to possess many of the same characteristics of DMBA in terms of its immunotoxicity.

Alpha Particles Induce Apoptosis through the Sphingomyelin Pathway

The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET α particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with α particles emitted by the ²²⁵Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated α particles using a planar ²⁴¹Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that α particles can activate the sphingomyelin pathway to induce apoptosis.

Abstract 5557: Steady DNA adduct accumulation by dibenzo[a,l]pyrene implants

Abstract The past and current trends in determining the DNA-damaging and carcinogenicity potential of environmental toxicants rely largely on treatment of rodents with single or multiple bolus doses. To simulate the human scenario where the exposure is generally continuous to low doses, we have developed polymeric implants embedded with carcinogens. When grafted subcutaneously, polymeric implants provide continuous (“24/7”) low dose exposure. In this study we report the effects of sustained low doses of a highly potent environmental carcinogen, dibenzo[a,l]pyrene (DBP) on DNA adduct accumulation. Different size DBP implants (10 – 20 mm long, 1.4 – 2.4 mm dia; 5% DBP load) were tested first in a pilot study using female A/J mice to optimize the implant surface area. Two 20-mm implants (1.4 mm dia), which produced maximal lung DNA adducts, were used subsequently. Groups of female A/J mice (n = 4 – 6) were grafted with sham or DBP implants. For comparison, animals were also treated in parallel with a bolus carcinogenic dose of DBP intraperitoneally (6 mg/kg) or vehicle alone. Animals from both the treatment groups were euthanized at different time intervals and selected tissues were collected. Analysis of the target (lung) DNA adducts by 32P-postlabeling showed qualitative identical adduct patterns by the two routes of administration; the sham treatments showed no detectable adducts. As expected, the bolus dose treatment resulted in higher DNA adducts (in adducts /109 nucleotides) (72 ± 18) compared with the implant group (33 ± 17) one week post-treatment. However, 3 weeks later, adduct levels in the implant group increased (54 ± 17) but a significant declined occurred in the i.p treatment group (19 ± 13). The adduct levels after 4 weeks in the implant group (66 ± 19) were somewhat higher than the 3 week group, suggesting that the adduct levels plateaued. The steady accumulation of DNA adducts was consistent with continuous release of DBP from the implant (about 10 µg/day based on in vitro release). The study is ongoing to determine if the low-dose continuous exposure which provides steady accumulation of DNA adducts in the target tissue will be tumorigenic. In summary, the steady-state adduct level reflect human exposure scenario and this approach will be ideal to determine both carcinogenicity and biological/pathological changes due to low-dose exposure as well as for screening potential chemopreventive agents. This exposure system has been used to determine efficacy of chemopreventive agents (accompanying abstracts by Aqil et al.; Gupta et al.) (Supported from CA-118114, KLCRP and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5557. doi:10.1158/1538-7445.AM2011-5557

A UVB Wavelength Dependency for Local Suppression of Recall Immunity in Humans Demonstrates a Peak at 300 nm

UVB radiation is a potent environmental carcinogen that not only causes mutations in the skin but also profoundly suppresses skin immune responses. Although this UVB-induced suppression of antitumor immunity has a key role in skin cancer development, the wavelengths within UVB causing greatest in vivo immunosuppression in humans are as yet unknown. We have identified a wavelength dependency for immunosuppression in humans across the UVB spectrum. We established linear dose-response curves for UV-induced local suppression of recall contact hypersensitivity responses at four wavelengths between 289 and 322 nm and found peak immune suppressive effectiveness at 300 nm and no detectable suppression at 322 nm within a physiologically relevant dose range.

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B(1), a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 A crystal structure displays an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 A crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs.

Histological evaluation of insulin receptors in mice kidneys and muscles in response to arsenic exposure.

Event Abstract Back to Event Histological evaluation of insulin receptors in mice kidneys and muscles in response to arsenic exposure. Theodora Papamitsou1, Ioanna Maggana1, Ioannis Tsomidis1, Ioannis Lialias1, Louisa Economou1 and Antonia Sioga1* 1 Aristotle University of Thessaloniki, Department of Histology and Embryology, Greece Introduction. Arsenic is one of the most potent environmental carcinogens. However, chronic exposure to the element or its compounds has also been associated with various non-cancerous diseases, including diabetes mellitus, especially non-insulin-dependent diabetes. Although mechanisms by which arsenic induces diabetes have not been identified, it seems that arsenic or its metabolites interfere with insulin-stimulated signal transduction pathway or with critical steps in glucose metabolism. The aim of this in vivo study was to correlate type 2 diabetes development with chronic arsenic exposure and to investigate the distribution of insulin receptors in kidneys and muscles. Methods. Fourteen Balb/c mice were divided into two groups: the first group was chronically exposed to arsenic (50 ppm) in drinking water, whereas the second group consumed distilled water. After the end of the 6 week period, animals were sacrificed and the kidneys and sciatic muscles were prepared for immunohistocemical analysis. Results. All animals in the arsenic group presented fasting hyperglycemia.The weight gain in both groups was not statistically significant. The immunohistochemical staining revealed significant decrease in the number of insulin receptors in both kidneys and sciatic muscles of the arsenic exposed mice. Conclusion. Our results have shown that prolong drinking of arsenic-contaminated water is associated with type 2 diabetes induction. Morphological and immunohistochemical changes caused by arsenic predominate in organs like kidneys and skeletal muscles.

Monitoring the Conformation of Benzo[a]pyrene Adducts in the Polymerase Active Site Using Fluorescence Resonance Energy Transfer

Benzo[a]pyrene (B[a]P) is a potent environmental carcinogen that is metabolized into diol epoxides that react with exocyclic amines in DNA. These DNA adducts have been shown to block DNA replication by high-fidelity polymerases and induce both base substitution and frame-shift mutations. To improve our understanding of the molecular mechanism of B[a]P-induced mutagenesis, a fluorescence resonance energy transfer (FRET) method was developed in which the (+)- or (-)-trans-anti-B[a]P-N(2)-dG adducts, positioned in the active site of DNA polymerase I (Klenow fragment), serve as donor fluorophores to an acceptor molecule positioned on the DNA primer strand. FRET was measured for a primer that ended one nucleotide before the adduct position and one that ended across from the adduct and used to estimate the distances between the two fluorophores. These estimates are consistent with prior studies that suggest the adducts are positioned in the minor groove. A comparison of the FRET for the (+)- and (-)-trans-B[a]P adducts in the Klenow active site suggested that the (+)-trans adduct is positioned approximately 2 A farther from the acceptor, consistent with the structural differences observed in duplex DNA where it has been shown that the (+)-trans adduct is oriented toward the 5'-end of the template strand while the (-)-trans adduct lies toward the 3'-end. Surprisingly, the adduct position did not change significantly when the primer was one nucleotide longer. The addition of either a correct (dCTP) or incorrect nucleotides showed only minor differences in FRET, suggesting that the adduct did not undergo a large change in the position within the polymerase active site, as expected if the adduct inhibited the polymerase conformational change.

Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay

A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha- ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay.