Gabriel W. Intano

Base Excision Repair Is Limited by Different Proteins in Male Germ Cell Nuclear Extracts Prepared from Young and Old Mice

ABSTRACT The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.

Spontaneous hepatocellular carcinoma is reduced in transgenic mice overexpressing human O6- methylguanine-DNA methyltransferase.

O6-methylguanine (O6mG) is a potent mutagenic and procarcinogenic DNA lesion. Organisms have evolved with a DNA repair mechanism that largely ameliorates the deleterious effects of O6mG through a direct reversal mechanism by a protein termed O6-methylguanine-DNA methyltransferase (MGMT). However, the contribution of O6mG to carcinogenesis, in the absence of known exposure to agents that produce it, has not been defined. Nontransgenic C3HeB male mice have a high frequency of spontaneous liver tumors. Transgenic CeHeB/FeJ mice expressing human MGMT (hMGMT) were generated that had elevated hepatic MGMT activity. The spontaneous development of hepatocellular carcinoma was significantly reduced in those mice expressing hMGMT compared with nontransgenic C3HeB/FeJ male mice. No differences were detected in spontaneous mutant frequencies in lacI transgenes in mice carrying hMGMT compared with that without hMGMT but the proportion of GC to AT transition mutations was lower in the transgenic mice carrying hMGMT as well as lacI. Tumors that arose in C3HeB/FeJ transgenic mice were largely deficient in hMGMT protein as determined by immunohistochemistry with a monoclonal antibody directed against hMGMT. Together these data indicate that spontaneous O6mG lesions induced hepatocellular carcinogenesis in C3HeB/FeJ male mice. These transgenic mice represent a rare example of reduced spontaneous carcinogenesis.

Health Span and Life Span in Transgenic Mice with Modulated DNA Repair

Abstract: One way to better understand the contribution of DNA repair, DNA damage, and mutagenesis in aging would be to enhance DNA repair activity, lower DNA damage, and lower mutagenesis. Because the repair protein O6‐methylguanine‐DNA methyltransferase (MGMT) acts alone and stoichiometrically, the human MGMT (hMGMT) cDNA was selected to test the feasibility of enhancing DNA repair activity in transgenic mice. MGMT activity is largely responsible for ameliorating the deleterious effects of O6‐methylguanine (O6mG) lesions in DNA in a direct reversal mechanism. A transgene was constructed consisting of a portion of the human transferrin (TF) promoter and hMGMT cDNA such that hMGMT is expressed in transgenic mouse brain and liver. Expression of hMGMT was associated with a significant reduction in the occurrence of an age‐related hepatocellular carcinoma in male mice at 15 months of age. Longitudinal and cross‐sectional studies were initiated to determine whether the reduced incidence of hepatocellular carcinoma would impact median or maximum life span. The cross‐sectional study performed on 15‐month‐old male animals confirmed the reduced occurrence of spontaneous hepatocellular carcinoma. At 30 months of age, however, the occurrence of hepatocellular carcinoma in at least one transgenic line was similar to that for nontransgenic animals. The longitudinal study is ongoing; however, at present no significant differences in life span have been detected. Tissues expressing the MGMT transgene also displayed greater resistance to alkylation‐induced tumor formation. These results suggest that transgenes can be used to direct enhanced DNA repair gene expression and that enhanced expression can protect animals from certain spontaneous and induced tumors.

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.

Relative Base Excision Repair in Xiphophorus Fish Tissue Extracts

Abstract: To begin characterizing DNA repair capability among Xiphophorus species, we adapted oligonucleotide-based DNA repair assays to extracts of fish tissues. Here, we report the initial results of relative base excision repair (BER) capability among 3 inbred Xiphophorus fish lines representing 2 species (X. maculatus and X. couchianus), and interspecies hybrid F1 animals produced by crossing them. Overall, data from uracil-N-glycosylase (UNG)–initiated BER assay (UNG-BER) indicate that brain tissue extracts generally possess higher BER activity than do gill and liver extracts. UNG-BER activities in gill and liver extracts were similar. The BER activity in the tissues of F1 interspecies hybrids followed the activity pattern of the X. couchianus parent in gill and liver extracts, was similar to the X. maculatus parent in brain extracts, but exhibited a reduced repair capacity in gill tissue extracts compared with either parent. We discuss the use of oligonucleotide-based DNA repair assays to elucidate the role that inheritance of DNA repair potential may play in susceptibility to disease and tumorigenesis in the intact organism.

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

Humans are exposed to ionizing radiation (IR) under various circumstances, e.g. cosmic radiation, diagnostic X-rays and radiotherapy for cancer. It has been shown that IR can impair spermatogenesis and can cause mutations in germ cells. However, the mutagenic responses of germ cells exposed to IR at different stages of testicular maturation have not been examined by directly assessing the mutant frequency in defined spermatogenic cell types. This study was performed to address whether preadult exposure to IR can increase mutations in adult germ cells that could in turn have a major impact on adult reproductive function and the health of ensuing offspring. Male Lac I transgenic mice were irradiated with a single dose of 2.5 Gy of gamma-ray at different ages before adulthood, reflecting different stages of testicular maturation, and then mutant frequency (MF) was determined directly in spermatogenic cell types emanating from the irradiated precursor cells. The results showed that (1) preadult exposure to IR did not significantly increase MF in adult epididymal spermatozoa; (2) spermatogenic stages immediately following the irradiated stage(s) displayed an elevated mutant frequency; but (3) the mutant frequency was restored to unirradiated levels in later stages of spermatogenesis. These findings provide evidence that there is a mechanism(s) to prevent spermatogenic cells with elevated mutant frequencies from progressing through spermatogenesis.