Hippocampal tissue of patients with refractory temporal lobe epilepsy is associated with astrocyte activation, inflammation, and altered expression of channels and receptors.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Previous research has demonstrated several trends in human tissue that, undoubtedly, contribute to the development and progression of TLE. In this study we examined resected human hippocampus tissue for a variety of changes including gliosis that might contribute to the development and presentation of TLE. The study subjects consisted of six TLE patients and three sudden-death controls. Clinicopathological characteristics were evaluated by H&E staining. Immunohistological staining and Western blotting methods were used to analyze the samples. Neuronal hypertrophy was observed in resected epileptic tissue. Immunohistological staining demonstrated that activation of astrocytes was significantly increased in epileptic tissue as compared to corresponding regions of the control group. The Western blot data also showed increased CX43 and AQP4 in the hippocampus and downregulation of Kir4.1, α-syntrophin, and dystrophin, the key constituents of AQP4 multi-molecular complex. These tissues also demonstrated changes in inflammatory factors (COX-2, TGF-ß, NF-κB) suggesting that these molecules may play an important role in TLE pathogenesis. In addition we detected increases in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and kainic acid receptor subunits KA1 (Grik4) and KA2 (Grik5) in patients' hippocampi. We noted increased expression of the α1c subunit comprising class C L-type Ca(2+) channels and calpain expression in these tissues, suggesting that these subunits might have an integral role in TLE pathogenesis. These changes found in the resected tissue suggest that they may contribute to TLE and that the kainic acid receptor (KAR) and deregulation of GluR2 receptor may play an important role in TLE development and disease course. This study identifies alterations in number of commonly studied molecular targets associated with astrogliosis, cellular hypertrophy, water homeostasis, inflammation, and modulation of excitatory neurotransmission in hippocampal tissues from TLE patients.

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.

A single-site mutation in the XPAC gene alters photoproduct recognition.

The XPAC (xeroderma pigmentosum group A complementing) gene, which is located on chromosome 9, carries a variety of point mutations in XP group A patients. We investigated the role of the XPAC gene product in excision repair by generating revertants of an XP group A cell line (XP12RO) that have increased resistance to ultraviolet light. One of these cell lines, XP129, can repair (6-4) pyrimidine-pyrimidone photoproducts normally but has reduced repair of cyclobutane dimers, as in XP12RO. Sequence analysis of cDNA from the XPAC gene indicated that XP12RO contains a termination codon at amino acid position 207, resulting in a reduced amount of mRNA and no detectable protein. In the revertant XP129 line, this termination codon has been mutated further and now encodes glycine in one allele instead of the wild-type arginine. The mRNA level detected by allele-specific polymerase chain reaction amplification was greater for the reverted sequence than for the chain-terminating sequence. These observations indicated that a point mutation resulting in a mis-sense mutation in the XPAC gene and altered expression of the XPAC protein can alter the substrate specificity of the excision repair system, and imply that the XPAC gene product plays an important role in photoproduct recognition.

Molecular dosimetry of DNA adducts and sister chromatid exchanges in human lymphocytes treated with benzo[a]pyrene.

We examined the relationship between benzo[a]pyrene-DNA adducts and sister chromatid exchanges (SCEs) in human lymphocytes. Cultures of isolated phytohemagglutinin (PHA)-stimulated lymphocytes from two normal donors were treated with 0.01-5.0 microM B[a]P from 24 to 72 h of culture. Using the highly sensitive 32P-postlabeling assay, we identified seven B[a]P-DNA adducts, one of which accounted for greater than 90% of the total DNA modifications. This adduct comigrated on polyethylenimine plates with the adduct produced by (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydro-benzo[a]pyrene. B[a]P-DNA adduct levels ranged from 0.02 to 8 adducts/10(7) nucleotides. SCE frequencies measured in parallel cultures ranged from 8 to 46 SCEs/cell. At the same B[a]P concentrations, B[a]P-induced SCE frequencies and B[a]P-DNA adduct levels were higher in lymphocytes from donor 1 than in lymphocytes from donor 2. There was a linear correlation between the number of B[a]P-DNA adducts and the number of SCEs induced; slopes of the linear regressions of induced SCEs on B[a]P-DNA adducts were similar for both donors. Our data suggest that SCE induction by B[a]P in human lymphocytes results from covalent DNA modification.