Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant.

The xeroderma pigmentosum (XP) variant (XPV) is a form of XP that has normal excision repair but shows defective DNA replication after UV irradiation. In developing various transformed fibroblast cell lines from these patients, we have found that there are significant phenotypic changes in transformed cells that seem to correlate with inactivation of p53. After transformation with SV40, XPV cell lines are only slightly UV sensitive, like their primary counterparts, but their sensitization with caffeine and the induction of sister chromatid exchanges (SCEs) by UV irradiation are greatly enhanced. After transformation by HPV16 E7, which targets the retinoblastoma cell cycle regulatory gene, there is no change in the UV sensitivity of XPV cells; but, when transformed by HPV16 E6 or E6 and E7 combined, there is a large increase in UV sensitivity and in the induction of SCEs. These changes are not associated with any detectable changes in the reactivation of an externally irradiated luciferase expression vector, the excision of cyclobutane pyrimidine dimers from bulk DNA, or unscheduled DNA synthesis and, therefore, do not involve excision repair. We suggest that if SCEs represent homologous recombination between sister chromatids, then in the absence of p53 function, the DNA chain arrest typical of UV-damaged XPV cells initiates strand exchange during recovery. In untransformed cells with normal p53, the preferred mode of recovery would then be replication bypass. The symptoms of elevated solar carcinogenesis in XPV patients may, therefore, be associated with increased genomic instability in cells of the skin in which p53 is inactivated by UV-induced mutations.

Overexpression of the XPA repair gene increases resistance to ultraviolet radiation in human cells by selective repair of DNA damage.

Overexpression of XPA genes, both wild type and a missense mutant, which code for a damage-specific, DNA-binding protein, increased the survival of repair-deficient and -competent human cells to levels above that of normal cells that did not overexpress XPA. The first 3 h after cells were damaged were most critical to achieving this increased survival. The dose at which 37% of the irradiated population survives could be restored to about one-half that of normal cells, with no detectable genome-wide repair of pyrimidine dimers or (6-4) photoproducts, suggesting that intermediate levels of XPA gene expression can direct repair to restricted critical regions of the genome. Current views of repair implicate transcriptionally active genes as a major component of such critical regions. Consistent with this interpretation, the repair of a transfected, actively expressed luciferase gene was higher than that of genomic DNA at intermediate and higher levels of XPA expression. High levels of XPA expression resulted in increased repair at early times after irradiation and extensive repair of (6-4) photoproducts but little, if any, pyrimidine dimer repair in the whole genome. At the highest level of expression, some clonal cell lines acquired resistance to radiation that corresponded to a dose at which 37% of the irradiated population survives that was about 1.5 to 2 times that of normal cells. The XPA gene product, therefore, can influence levels of DNA repair and radiation sensitivity quantitatively by contributing to selective repair at certain sites in the genome.

Genetic and molecular ecotoxicology: a research framework.

Participants at the Napa Conference on Genetic and Molecular Ecotoxicology assessed the status of this field in light of heightened concerns about the genetic effects of exposure to hazardous substances and recent advancements in our capabilities to measure those effects. We present here a synthesis of the ideas discussed throughout the conference, including definitions of important concepts in the field and critical research needs and opportunities. While there were many opinions expressed on these topics, there was general agreement that there are substantive new opportunities to improve the impact of genetic and molecular ecotoxicology on prediction of sublethal effects of exposure to hazardous substances. Future studies should emphasize integration of genetic ecotoxicology, ecological genetics, and molecular biology and should be directed toward improving our understanding of the ecological implications of genotoxic responses. Ecological implications may be assessed at either the population or ecosystem level; however, a population-level focus may be most pragmatic. Recent technical advancements in measuring genetic and molecular responses to toxicant exposure will spur rapid progress. These new techniques have considerable promise for increasing our understanding of both mechanisms of toxicity on genes or gene products and the relevance of detrimental effects to individual fitness.

Effects of acute and chronic acidification on three larval amphibians that breed in temporary ponds.

This study explored the effects of acute (7 days) and chronic (4 months) exposure to pH 4.2 on three species of larval amphibians, Ambystoma jeffersonianum, Ambystoma maculatum, and Rana sylvatica. Acute tests were conducted in 24 impermeable enclosures in three temporary ponds. Total dissolved aluminum was higher in acidified enclosures in comparison with controls (pH 4.2, [Al] approximately 10-30 microM and pH greater than 4.7, [Al] approximately 5-15 microM, respectively). Greater mortality of A. jeffersonianum occurred at pH 4.2 than at pH greater than 4.7, whereas survival of A. maculatum and R. sylvatica were unaffected by pH. Mean wet masses of R. sylvatica were significantly lower at pH 4.2 than at pH greater than 4.7, but mean wet masses of surviving A. jeffersonianum and A. maculatum were not influenced by pH. There were no pH-related differences in body sodium concentration in larval R. sylvatica. Chronic acidification of mesocosms to pH 4.2 ([Al] approximately 16 microM) (controls = pH greater than 6, [Al] approximately 0.1 microM) resulted in total mortality of A. jeffersonianum. Survival of A. maculatum and R. sylvatica were not associated with pH, but survival of A. maculatum was low at both pH levels. Time to metamorphosis was longer for R. sylvatica maintained at pH 4.2, but not for A. maculatum. No differences in wet masses at metamorphosis were observed for R. sylvatica or A. maculatum. These results indicate that short and long term acidification of temporary wetlands could dramatically affect amphibians which rely upon them as breeding sites, either by causing mortality or by decreasing growth rates.