Sequence Variant in the TRIM39-RPP21 Gene Readthrough is Shared Across a Cohort of Arabian Foals Diagnosed with Juvenile Idiopathic Epilepsy.

Juvenile idiopathic epilepsy (JIE) is a self-limiting neurological disorder with a suspected genetic predisposition affecting young Arabian foals of the Egyptian lineage. The condition is characterized by tonic-clonic seizures with intermittent post-ictal blindness, in which most incidents are sporadic and unrecognized. This study aimed to identify genetic components shared across a local cohort of Arabian foals diagnosed with JIE via a combined whole genome and targeted resequencing approach: Initial whole genome comparisons between a small cohort of nine diagnosed foals (cases) and 27 controls from other horse breeds identified variants uniquely shared amongst the case cohort. Further validation via targeted resequencing of these variants, that pertain to non-intergenic regions, on additional eleven case individuals revealed a single 19bp deletion coupled with a triple-C insertion (Δ19InsCCC) within the TRIM39-RPP21 gene readthrough that was uniquely shared across all case individuals, and absent from three additional Arabian controls. Furthermore, we have confirmed recent findings refuting potential linkage between JIE and other inherited diseases in the Arabian lineage, and refuted the potential linkage between JIE and genes predisposing a similar disorder in human newborns. This is the first study to report a genetic variant to be shared in a sub-population cohort of Arabian foals diagnosed with JIE. Further evaluation of the sensitivity and specificity of the Δ19InsCCC allele within additional cohorts of the Arabian horse is warranted in order to validate its credibility as a marker for JIE, and to ascertain whether it has been introduced into other horse breeds by Arabian ancestry.

Evaluation of genotoxic and cytotoxic properties of pesticides employed in Italian agricultural practices.

In a program coordinated by the Italian Ministry of Works, we tested in vitro four pesticides widely employed in a developed agricultural region of central Italy. The four commercial agents were chosen on the basis of their diffusion in agricultural practice, knowledge of their active principle(s), and scant availability of data concerning their toxic and genotoxic activity. The agents were Cirtoxin, Decis, Tramat Combi (TC), and Lasso Micromix (LM). All substances were tested in three in vitro systems: Chinese hamster ovary (CHO) cells, a metabolically competent hamster cell line (Chinese hamster epithelial liver; CHEL), and root tips of Vicia faba (VF). The cytotoxic and genotoxic end points challenged were micronuclei and root tip length (RTL) in VF and mitotic index (MI), proliferation index (PI), cell survival (CS), cell growth (CG), cell cycle length (CCL), sister chromatid exchanges, chromosomal aberrations, and single-cell gel electrophoresis, or comet assay, in CHEL and CHO cells. Tested doses ranged from the field dose up to 200x the field dose to take into account accumulation effects. On the whole, tested agents appear to induce genotoxic damage only at subtoxic or toxic doses, indicating a low clastogenic risk. MI, PI, CS, CG, RTL, and CCL appear to be the less sensitive end points, showing no effects in the presence of a clear positive response in some or all of the other tests. Using cytogenetic tests, we obtained positive results for TC and LM treatments in CHO but not in CHEL cells. These data could be accounted for by postulating a detoxifying activity exerted by this cell line. However, cytogenetic end points appear to be more sensitive than those referring to cytotoxicity.

DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro.

Among other targets, DNA demethylating agents are known to affect the sister chromatid exchange (SCE) frequency in mammalian cells in vitro. The SCE increase appears to be maintained for many (10-16) cell cycles after the end of the pulse in a given cell population, unlike SCEs induced by DNA damaging agents. Yet, epigenetic changes (such as demethylation) would not be expected to affect SCE at all. In the present report we challenge the working hypothesis of a relation between SCEs and demethylation by comparing SCE induction during different rounds of replication when the parental strands were normally methylated or demethylated. Azacytidine (AZA), ethionine (ETH), mitomycin-C (MMC), UV-irradiation (UV) and hydrogen peroxide (H(2)O(2)) were tested for SCE induction in a Chinese hamster ovary cell line after a single pulse, one or two cell cycles before fixation. Whereas MMC, UV and H(2)O(2) induce SCE in both protocols, AZA and ETH show an effect on SCEs only if administered two cycles before fixation. Because two cell cycles are needed in order to achieve demethylation of the parental DNA strand, the data reported here support our working hypothesis that demethylation in the parental DNA strand, at the level of the replication fork (i.e., the region where SCEs are formed), is responsible for an increase in mistaken ligations of processed damage, eventually yielding an increase in SCEs.

Persistence of sister chromatid exchange by 9-beta-D-arabinofuranosyladenine in Chinese hamster ovary cells cultivated in vitro.

The cytogenetic activity of 9-beta-D-arabinofuranosyladenine (ara-A), a known antitumor agent with an unknown mechanism of action (possibly acting via demethylation and/or decrease in DNA synthesis), has been tested in Chinese hamster ovary (CHO) cells cultivated in vitro, with respect to its ability to induce sister chromatid exchange (SCE). The agent shows no effect on cell cycle parameters (proliferation and mitotic indices) at concentrations up to 100 microM, when administered in pulses ranging from 3 to 12 h. Furthermore, even if administered over the entire treatment period (24 h), a severe cell cycle delay appears only at a concentration of 100 microM. A clear increase in the SCE frequency is produced starting from a concentration of 10 microM, irrespective of the treatment protocol (i.e. pulse versus continuous). This effect on the SCE frequency is maintained for as long as 10 cell cycles after removal of the agent. A partial inactivation of S-adenosyl-L-homocysteine (AdoHcy) hydrolase seems to be produced for as long as 6 h after agent removal. We suggest that under our experimental conditions ara-A exerts its action through a heritable epigenetic modification.

Antimutagenic activities of naturally occurring polyamines in Chinese hamster ovary cells in vitro.

Spermine and spermidine, ubiquitous polyamines present in bacteria and animal cells, are also involved in cell growth. Since they interact with the double helix, they can stabilize the DNA molecule. Recent evidence of the antimutagenic and anticarcinogenic capacity of spermine has focused attention on the mechanism(s) by which such agents can protect cells from induced damages. In the present paper we show the ability of spermine and spermidine to decrease the level of sister chromatid exchanges induced in Chinese hamster ovary cells cultivated in vitro, by treating them with Psoralen + UVA irradiation (able to induce mainly monoadducts and DNA cross-links). Two different mechanisms of polyamine action can be invoked to explain the preservative activity of this class of agents.

Retinoids as modulators of metabolism: their inhibitory effect on cyclophosphamide and 7,12-dimethylbenz[a]anthracene induced sister chromatid exchanges in a metabolically competent cell line.

Recently, retinoids have been studied for their ability to modify the carcinogenic/mutagenic activity of chemical compounds. Results show that they can inhibit the malignant transformation of cells, the induction of cancer in experimental animals and the mutagenicity of promutagens. Our experiments examine how retinol and retinoic acid can decrease the frequency of sister chromatid exchange (SCE) induced by two indirect mutagens/carcinogens (cyclophosphamide and 7,12-dimethylbenz[a]anthracene) in an epithelial liver cell line of male Chinese hamster (CHEL cells). These cells are metabolically competent and activate different classes of promutagens into biologically active metabolites. Our results are consistent with the suggestion that retinoids modulate the genotoxicity of indirect-acting mutagens by altering their metabolic activation or cellular detoxification processes or both.

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells.

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.

Induction of chromosome aberrations by chemical mutagens in neural ganglia of Drosophila melanogaster.

Chromatid aberrations induced by various concentrations of bleomycin, cyclophosphamide and mitomycin C were analyzed in neural ganglia of third-instar larvae of Drosophila melanogaster. A clear dose response was observed with increasing dose after treatment with bleomycin and mitomycin C, whereas no effect was observed after treatment with cyclophosphamide. A comparison with published data for the induction of sex-linked recessive lethals showed that, at least for the 3 drugs tested, the use of both tests eliminates false negatives and might comprise a useful procedure for testing mutagenicity in Drosophila.

Cell-stage-specific enhancement by caffeine of the frequency of chromatid aberrations induced by X-rays in neural ganglia of Drosophila melanogaster.

Caffeine (10(-2) M) induced a high level of chromatid aberrations in neural ganglia of third-instar larvae of Drosophila melanogaster only when it was added to cells in late G2 and mitotic prophase. No aberrations were observed after treatment in late S--middle G2 or C-mitosis. We observed that, in these stages, caffeine strongly increased X-ray-induced damage (500 R). This potentiation was quantitatively similar. But it involved all types of aberration after treatment in C-mitosis, and essentially isochromatid deletions and chromatid exchanges after treatment in S--G2. Some hypotheses are put forth to explain the possible mechanism of action of caffeine in the potentiation of X-ray-induced damage.