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Intramural Papers of the Month

By Laura Hall and Angelika Zaremba
June 2010

Gene Expression-Based Predictive Model for Hepatocarcinogenicity

NIEHS scientists in the National Toxicology Program (NTP) have developed a gene expression-based predictive model to identify hepatocarcinogens.

The current high-sensitivity NTP protocol to test the toxicity and carcinogenicity of chemicals in two-year rodent bioassays is very expensive. Only a small fraction of the estimated 30,000 chemicals in widespread commercial use in the U.S. have been tested.

An accurate method that can screen chemicals to more rapidly identify potential human carcinogens is critically needed. To address this need, researchers with NTP developed pattern-recognition models based on rat liver gene expression changes induced by subchronic chemical exposure to differentiate hepatocarcinogens from non-hepatocarcinogens.

After training the models on gene expression elicited by a set of chemicals with known carcinogenic activity, the researchers were able to accurately predict the known hepatocarcinogenicity of an independent set of alkenylbenzene flavoring agents -- even predicting that two alkenylbenzenes never tested for carcinogenicity would be weakly hepatocarcinogenic in rat at a certain dose level. The study also showed that exposure duration is a critical variable and that the 90-day exposure data gave more accurate predictions.

Citation: Auerbach SS, Shah RR, Mav D, Smith CS, Walker NJ, Vallant MK, et al ( Exit NIEHS. 2010. Predicting the hepatocarcinogenic potential of alkenylbenzene flavoring agents using toxicogenomics and machine learning. Toxicol Appl Pharmacol 243(3):300-314.

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Periodontal Pathogen Infection Has a Potential Protective Effect in Asthma

NIEHS scientists demonstrated a connection between infection with the oral pathogen Porphyromonas gingivalis ( P. gingivalis) and a decrease in the lung inflammatory response to inhaled allergen - a phenomenon that may be important in the pathogenesis of asthma.

The team of researchers infected mice with P. gingivalis using a surgically implanted chamber. The mice were sensitized with the allergen ovalbumin before or after establishment of infection to study the effects of the infection on allergic airway inflammation and airway responsiveness, the two hallmarks of asthma. Oral pathogen infection prior to allergic sensitization led to decreased histological inflammation and reduced airway levels of the T-helper lymphocytes type 2 (Th2) cytokines interleukin-4, -5, -13, but had no effect on airway responsiveness.

In contrast, oral pathogen infection after allergic sensitization did not alter inflammatory endpoints, but led to reduced airway responsiveness. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness.

A prior study by the research group demonstrated an inverse relationship between antibodies to two oral pathogens and asthma, wheeze, and hay fever in humans enrolled in the National Health and Nutrition Examination Survey (NHANES).

Together, these findings are consistent with the hygiene hypothesis which contends that fewer opportunities for infection have led to increases in asthma and other allergic diseases.

Citation: Card JW, Carey MA, Voltz JW, Bradbury JA, Ferguson CD, Cohen EA, et al ( Exit NIEHS. 2010. Modulation of allergic airway inflammation by the oral pathogen Porphyromonas gingivalis. Infect Immun 78(6):2488-2496.

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Oxidation State Alters Binding Affinity of Scaffolding Protein Involved in DNA Repair

The repair of DNA lesions requires multiple enzymes whose activities are organized by scaffolding proteins. One such protein, X-ray repair cross complementing group 1 (XRCC1), plays important roles in both base excision repair (BER) and single strand break repair. NIEHS scientists have structurally characterized the molecular interface connecting the XRCC1 N-terminal domain (XRCC1-NTD) and the catalytic domain of a DNA polymerase, polymerase beta (Pol beta), that form part of the repair complex.

The scientists discovered that XRCC1 can exist in two different forms -- a reduced structure observed previously, as well as an oxidized structure. The oxidized form has substantial changes in secondary structure, folding topology, and electrostatic surface associated with the formation of a disulfide bond.

The results showed that although most of the structural changes are not located on the Pol beta binding interface, the interaction was sufficiently altered so that the affinity of oxidized XRCC1 for Pol beta was increased by an order of magnitude.

The researchers note that oxidation-dependent affinity of XRCC1 for Pol beta is consistent with the response of BER to oxidative stress. They suggest that the redox state of scaffolding proteins plays an important role in DNA repair by causing changes in the structure of the repair complex.

Citation: Cuneo MJ, London RE ( Exit NIEHS. 2010. Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity. Proc Natl Acad Sci U S A 107(15):6805-6810.

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Novel Statistical Method for Testing Haplotype-Environment Interactions

NIEHS researchers developed a novel statistical method to study multiplicative interactions of many single nucleotide polymorphisms (SNPs) simultaneously in relation to an environmental exposure. In simulations, this new test respects the nominal Type 1 error rate, provides good power under a variety of scenarios, and appears resistant to exposure-related population stratification bias.

SNP variations of a gene's DNA sequence can cause functional differences in its protein product and may contribute to disease. A haplotype is a set of SNPs that are close together on the chromatid, making them likely to be inherited together and, thereby, statistically associated. The analysis of haplotype-exposure interactions is important for investigating the causes of common diseases.

The method uses families consisting of an affected offspring (either exposed or not) and two biological parents. Genetic variants that increase susceptibility will tend to have been transmitted too often to affected offspring. If the genetic variant and the exposure work synergistically to cause the disease, this tendency will be more pronounced among the exposed than the unexposed.

The new non-parametric statistical method is reasonably powerful, handles missing genotypes, retains validity under any genetic main effects, tolerates Hardy-Weinberg disequilibrium, and does not require the user to know or estimate candidate haplotypes.

Citation: Shi M, Umbach DM, Weinberg CR ( Exit NIEHS. 2010. Testing haplotype-environment interactions using case-parent triads. Hum Hered 70(1):23-33.

(Laura Hall is a biologist in the NIEHS Laboratory of Toxicology and Pharmacology currently on detail as a writer for the Environmental Factor. Angelika Zaremba, Ph.D., is a visiting postdoctoral fellow in the NIEHS Laboratory of Signal Transduction Inositol Signaling Group.)

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