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Intramural Papers of the Month

By Robin Arnette
July 2008

Exposure to Pesticides Increases Risk of Diabetes

Exposure to certain pesticides increased the risk of diabetes in licensed applicators, according to researchers from NIEHS and the National Cancer Institute. The investigation of 33,457 applicators enrolled in the Agricultural Health Study (AHS) is the largest study to date to evaluate potential effects of pesticides on diabetes incidence in adults.

Because previous cross-sectional studies using data from the National Health and Nutrition Examination Survey (NHANES) found associations of diabetes with serum levels of persistent organic pollutants, the researchers wanted to know if there was a similar association between diabetes and lifetime exposure to pesticides. Therefore, they evaluated applicators who reported diabetes for the first time in five-year follow-up telephone interviews, conducted between 1999 and 2003. Previously, at enrollment in 1993 to 1997, applicators had described use of 50 different pesticides, providing information on two primary measures: ever use and cumulative lifetime days of use.

Of 50 pesticides evaluated, seven were associated with an increased incidence of diabetes using both exposure measures. Three of these were organochlorine insecticides (aldrin, chlordane, heptachlor), two were organophosphate insecticides (trichlorfon, dichlorvos), and two were herbicides (alachlor, cyanazine). The strongest association was with trichlorfon: applicators that had used the chemical on more than 10 days in their lifetime had a 2.5-fold increase in risk.

Citation: Montgomery MP, Kamel F, Saldana TM, Alavanja MC, Sandler DP( Exit NIEHS Website. 2008. Incident diabetes and pesticide exposure among licensed pesticide applicators: Agricultural Health Study, 1993-2003. Am J Epidemiol 167(10):1235-1246.

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Sox8 Plays an Important Role in Male Fertility

Although the transcription factor SOX8 isn't needed during testis development, it is necessary for the maintenance of adult male fertility. The finding came from a collaborative team made up of scientists from NIEHS, the Monash Institute of Medical Research, ARC Centre of Excellence in Biotechnology and Development, the University of Queensland and Merck Research Laboratories. This study provides a framework to determine if SOX8 mutations are involved in the low sperm counts seen in men.

The team generated a strain of Sox8 knockout mice (Sox8-/-) and compared them to heterozygous (Sox8+/-) and wild-type control male mice (Sox8+/+). Sperm motility was measured at two and five months, while total body weight and testicular weight were measured at two, five and nine months. Testicular tissue was subjected to immunohistochemical staining using Espin and Vinculin, two proteins involved in cell adhesion.

The data demonstrated that loss of SOX8 disturbed the interaction between Sertoli cells and the developing germ cells. This action led to the progressive degeneration of the seminiferous epithelium and decreased activity beyond the first wave of spermatogenesis.

Citation: O'Bryan MK, Takada S, Kennedy CL, Scott G, Harada S, Ray MK, Dai Q, Wilhelm D, de Kretser DM, Eddy EM, Koopman P, Mishina Y.( Exit NIEHS Website 2008. Sox8 is a critical regulator of adult Sertoli cell function and male fertility. Dev Biol 316(2):359-370.

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The Involvement of DNA Polymerases in Eukaryotic Replication

For several decades, researchers weren't sure which DNA polymerase copied the leading and lagging strand templates during eukaryotic chromosomal replication. Using the replication fork in Saccharomyces cerevisiae, scientists from NIEHS and Washington University School of Medicine have clarified this question by determining that under normal conditions, DNA polymerase δ (Pol δ) copies the lagging strand while DNA polymerase e (Pol e) copies the leading strand. The results suggest a nearly equal strand-specific division of labor between the two polymerases.

The team used a mutant Pol δ allele (L612M) in a yeast genetic system. This mutant was chosen because its error rate is higher for one mismatch, for example T•dGTP, than for its complement A•dCTP, enabling the researchers to assign mutations generated by L612M Pol δ to either the leading or lagging strand. L612M Pol δ mutagenesis was dependent on the orientation of a reporter gene relative to an adjacent replication origin, indicating strand specificity. The identity of the preferentially targeted strand was revealed using the biased error rates of L612M Pol δ. The results imply that greater than 90 percent of L612M Pol δ synthesis was performed using the lagging strand as a template rather than the leading strand.

The researchers also investigated mismatch repair efficiency and intend to further this work by determining if the division of labor varies under different cellular conditions, such as after the replication fork stalls or is blocked.

Citation: Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM, Kunkel TA.( Exit NIEHS Website 2008. Division of labor at the eukaryotic replication fork. Mol Cell 30(2):137-144.

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Structures of DNA Polymerase β Provide the First Glimpse of Pre-Mutagenic DNA Synthesis

DNA polymerases read the nucleotide sequence of a "template" strand of DNA as they synthesize a new complementary strand of DNA-obeying Watson-Crick base pairing rules. The synthesis process is highly accurate, but occasionally a polymerase will make an error and insert an incorrect nucleotide into the new DNA strand. This action can lead to an alteration in the genetic material termed a "mutation."  Some mutations are known to be very important in human disease and in conditions such as aging, but insight on how mutations occur during DNA synthesis has remained a mystery.

Researchers at NIEHS used crystallographic structures of DNA polymerase β (Pol β) with right (matched) and wrong (mismatched) nucleotide substrates to gain insight on how mutations are averted during the enzymatic process of DNA synthesis.  The team created G-A and C-A mismatches in the Pol b active site by employing a stable nucleotide analog, dAMPCPP, which could bind to the polymerase but not be inserted.  Additionally, it was necessary to substitute manganese for magnesium, a divalent metal necessary for DNA synthesis. Kinetic assays revealed that manganese could substitute for magnesium, and increased the binding affinity for the wrong nucleotide to form the pre-requisite ternary complex (Pol β/DNA/nucleotide) crystal.

Surprisingly, the structures revealed that both types of substrates (matched and mismatched) produced the same polymerase conformation. However, the mismatched substrate induced a shift in the template strand that produced an abasic site-like pre-synthesis intermediate. The structures are consistent with mutagenesis studies and provide a strategy to avert misinsertion of the wrong nucleotide. This study sheds light on the specific structural changes necessary during high fidelity DNA synthesis, a process central to DNA repair and replication.

Citation: Batra VK, Beard WA, Shock DD, Pedersen LC, Wilson SH.( Exit NIEHS Website Structures of DNA polymerase beta with active site mismatches suggest a transient abasic site intermediate during misincorporation. Mol Cell 2008 May;30(3):315-324.

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