Trichinocidal activity of a novel formulation of curcumin-olive oil nanocomposite in vitro.

Curcumin-olive oil nanocomposite (CO-NC), a novel formulation of nano-curcumin, was produced and characterized. By evaluating the death rate and DNA damage inflicted on adult Trichinella spiralis (T. spiralis) worms using the comet test and Scanning electron microscopy (SEM) analysis, the effectiveness of the substance against these worms was assessed in vitro. The mortality effects of CO-NC on the parasite adult worms were increased with the upgrading in the concentration and exposure time from 1 to 24 h using concentrations from 10 to 100 ppm. LC50 was determined to be 10.0 ppm/18 h, 20.0 ppm/9 h, 40.0 ppm/6 h, 80.0 ppm/2 h, and 100.0 ppm/1 h, while LC100 was 40.0 ppm/24 h, 80.0 ppm/12 h, and 100.0 ppm/6 h. The comet assay was utilized to examine DNA damage in control and dead worms exposed to varying doses. A direct correlation (P ≤ 0.05) was found between the increase in CO-NC dose and the degree of DNA damage as indicated by alterations in DNA % in the tail segment, tail length (µm), tail moment (µm), and olive tail moment with the control samples. The sub-epidermal layer was detached, the cuticle was partially sloughed off, and the usual creases, ridges, and annulations were altered in the T. spiralis exposed worms. As a result, the tested new trichinocidal drug formulation of nano-curcumin on an oil base was confirmed to be an efficient, secure, and environmentally friendly alternative. The medication has the potential to severely and irreversibly harm the DNA and ultrastructural morphology of adult worms.

Amelioration potential of Moringa oleifera extracts against sodium arsenate induced embryotoxicity and genotoxicity in mouse (Mus musculus) / Potencial de melhoria de extratos de Moringa oleifera contra embriotoxicidade e genotoxicidade induzida por arseniato de sódio em camundongos (Mus musculus)

Previous studies have suggested that arsenic crosses the placenta and affects the fetus development. The study under consideration aims to show comparative ameliorative effect of Moringa oleifera leaf and flower extracts against sodium arsenate induced fetus toxicity of mice. Pregnant mice (N=44) were kept in lab and divided into eleven group from (A to K) and were orally administered the doses 6 mg/kg, 12 mg/kg for sodium arsenate, 150 mg/kg and 300 mg/kg for Moringa oleifera leaf extracts (MOLE) and 150 mg/kg and 300 mg/kg for Moringa oleifera flower extracts (MOFE) comparing with control. The investigation revealed evident reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumferences well as malformations in tail, feet, arms, legs, skin and eyes in the negative control group (only administered with sodium arsenate). Co-administration of sodium arsenate with MOLE and MOFE ameliorate the reversed effect of sodium arsenate on the shape, length, body weight and DNA damage of fetus significantly at 95% confidence interval. However, Moringa oleifera leaf extract showed more significant results in comparison to Moringa oleifera flower extract. Hence concluded that Moringa oleifera leaf extract ameliorated the embryo toxic effects of sodium arsenate and can be used against environmental teratogens.

Estudos anteriores sugeriram que o arsênio atravessa a placenta e afeta o desenvolvimento do feto. O estudo em consideração visa mostrar o efeito melhorador comparativo de extratos de folhas e flores de Moringa oleifera contra a toxicidade fetal induzida por arseniato de sódio em camundongos. Camundongos grávidas (N = 44) foram mantidos em laboratório e divididos em 11 grupos (de A a K) e foram administrados por via oral nas doses de 6 mg/kg, 12 mg/kg para arseniato de sódio, 150 mg/kg e 300 mg/kg para extratos de folhas de Moringa oleifera (MOLE) e 150 mg/kg e 300 mg/kg para extratos de flores de Moringa oleifera (MOFE) em comparação com o controle. A investigação revelou redução evidente no peso do feto, membro posterior, membro anterior, comprimento da cauda e focinho, coroa, nádega e circunferência da cabeça, bem como malformações na cauda, pés, braços, pernas, pele e olhos no grupo de controle negativo (apenas administrado com arseniato de sódio). A coadministração de arseniato de sódio com MOLE e MOFE melhora significativamente o efeito reverso do arseniato de sódio na forma, comprimento, peso corporal e dano ao DNA do feto, com intervalo de confiança de 95%. No entanto, o extrato da folha da Moringa oleifera apresentou resultados mais significativos em comparação ao extrato da flor da Moringa oleifera. Portanto, concluiu que o extrato da folha de Moringa oleifera melhorou os efeitos tóxicos do arseniato de sódio para o embrião e pode ser usado contra teratógenos ambientais.

Modified comet assay with Giemsa staining: a suitable method for assessing genotoxicity in rats / Ensaio cometa modificado com coloração de Giemsa: um método adequado para avaliar a genotoxicidade em ratos

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)

O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)

Genotoxicity assessment of fipronil (frontline plus®) in Canis familiaris / Avaliação de genotoxicidade do fipronil (frontline plus®) em cães

Fipronil is a pesticide widely used for controlling fleas and ticks in domestic animals, and its short-term exposure can lead to serious effects on animals. However, the possible genotoxic effect of this compound has not been investigated in target animals. Based on the hypothesis that fipronil can induce genotoxicity, this study evaluated the effect of fipronil on DNA damage in peripheral blood cells. For that purpose, ten dogs of both sexes were used in the study. The product (6.7mg/kg) was applied on the dorsal neck region of each animal. Peripheral blood samples were collected immediately prior to application of the product, and at 3, 8 and 24 hours after the application. Samples were processed for comet assay. No statistically significant differences were found among the four time points. The current study suggests for the first time that a single exposure to this pesticide does not induce systemic genotoxic effect in dogs.(AU)

O fipronil é um inseticida/herbicida amplamente utilizado para controle de pulgas e carrapatos em animais domésticos. Sua exposição a curto prazo tem acarretado efeitos deletérios em animais. Entretanto, o possível efeito genotóxico deste composto ainda não foi investigado em animais alvo. Baseando-se na hipótese de que o fipronil pode induzir genotoxicidade, o presente estudo avaliou o efeito deletério do fipronil no material genético de células de sangue periférico. Para isso, dez cães sadios, de ambos os sexos, foram utilizados neste estudo. O produto (6,7mg/kg) foi aplicado na região dorsal do pescoço de cada animal. As amostras de sangue foram coletadas imediatamente antes da aplicação do produto (controle) e após três, oito e 24 horas da aplicação. As amostras foram imediatamente processadas para condução do teste do cometa, a fim de se avaliar os danos basais no DNA. Não houve diferença significativa entre os quatro momentos de coleta em relação aos danos no material genético. O estudo sugere, pela primeira vez, que uma exposição única a este pesticida não induz efeito genotóxico sistêmico em cães.(AU)

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis.

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd2+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd2+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

Longitudinal profile of the genotoxic potential of the River Danube on erythrocytes of wild common bleak (Alburnus alburnus) assessed using the comet and micronucleus assay.

The Joint Danube Survey 3 (JDS3; the biggest river expedition in 2013) had offered the unique opportunity for a large-scale monitoring approach for biomarker response in feral fish collected along a Danube stretch from Kehlheim (DE) to Sulina (RO). The advantage of genotoxicity as a marker for pollution exposure in fish is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was in the focus of the biomarker investigations in fish during the expedition. Blood samples of common bleak (Alburnus alburnus) for the investigation of the micronucleus frequency and comet tail intensity of fragmented DNA material in erythrocytes were collected at 18 and 12 sampling sites, respectively. For 9 sampling sites same samples were used to compare the in-situ data for the comparable genotoxic endpoint in the micronucleus (MN) and comet assay (CM). The data of both in-situ assays showed a significant correlation, indicating the strength and comparability of the data sets. Significant variation in DNA damage in fish along the longitudinal profile of the Danube was demonstrated for both assays compared to reference sites. The results suggest that DNA damage in erythrocytes of fish was mainly affected by wastewater of highly populated regions. No linkage between the results and the general health/dietary status of the fish were revealed, whereas correlation with some genotoxicity drivers in the water phase, suspended particulate matter and sediments could be demonstrated.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd²+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd²+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (Cyprinus carpio).

Amino acids have an important biological role for prevention of cell damage during cryopreservation. The objective of this study is to determine the effects of cysteine on postthaw sperm motility, duration of sperm motility, DNA damage, and fertility in the common carp (Cyprinus carpio). Sperm collected from 10 individuals was cryopreserved in extenders containing different cysteine concentrations (2.5, 5, 10, and 20 mM). Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution, the semen was aspirated into 0.25-mL straws; the straws were placed on the tray, frozen in nitrogen vapor, and plunged into liquid nitrogen. DNA damage was evaluated by comet assay after cryopreservation. Our results indicated that an increase in the concentration of cysteine caused a significant increase in the motility rate and duration of sperm in the common carp (C carpio; P < 0.05). Comparing all concentrations of cysteine, the best concentration of cysteine was 20 mM. Higher postthaw motility (76.00 ± 1.00%) and fertilization (97.00 ± 1.73%) rates were obtained with the extender at the concentration of 20 mM. Supplementation of the extender with cysteine was increased the fertilization and hatching rate and decreased DNA damage. Consequently, cysteine affected the motility, fertilization, and DNA damage positively, and extenders could be supplemented with cysteine.

Acute copper toxicity in juvenile fat snook Centropomus parallelus (Teleostei: Centropomidae) in sea water

Three experiments were designed to assess the accumulation and acute toxicity of copper (Cu) in juvenile fat snook Centropomus parallelus. The first experiment was performed to determine the 96-h lethal concentration (LC50) of Cu. The second experiment was designed to assess the effects of sublethal concentrations of Cu (0.47 and 0.94 mg/L), while the third one allowed us to test the recovery capacity of fish exposed to the sublethal concentrations Cu and kept in sea water without Cu addition. The LC50 value for Cu was found to be 1.88 mg/L Cu. Fish exposed to the sublethal concentrations of Cu showed a significant accumulation of Cu in gills at 96 h respect to the control ones (0.43 µg/g Cu). No significant difference was observed in the accumulation of Cu in gills between fish exposed to 0.47 mg/L (1.09 µg/g Cu) and 0.94 mg/L (1.26 µg/g Cu). Exposure (24 and 96 h) to the sublethal concentrations of Cu tested induced DNA damage in the erythrocytes. The results show that acute exposure to sublethal concentrations induces Cu accumulation and DNA damage in fish, these effects being recovered after 240 h in sea water without Cu addition.

Três experimentos foram realizados para avaliar o acúmulo e toxicidade aguda do cobre (Cu) em juvenis de robalo-peva Centropomus parallelus. O primeiro experimento foi realizado para determinar a concentração letal (96h-CL50) de Cu. O segundo experimento foi realizado para avaliar os efeitos de concentrações subletais de Cu (0,47 e 0,94 mg/L), enquanto o terceiro permitiu testar a capacidade de recuperação dos peixes expostos a concentrações subletais do Cu e posteriormente mantidos em água do mar sem acréscimo de Cu. O valor de LC50 encontrado para o Cu foi de 1,88 mg/L. Os peixes expostos as concentrações subletais de Cu mostraram um acúmulo significativo nas brânquias em relação ao controle em 96 h de exposição (0,43 µg/g Cu). Nenhuma diferença significativa foi observada entre os peixes expostos a 0,47 mg/L de Cu (1,09 µg/g) e 0,94 mg/L de Cu (1,26 µg/g). A exposição (24 e 96 h) para as concentrações subletais de Cu induziram danos no DNA. Os resultados mostram que a exposição aguda a concentrações subletais induz o acúmulo de Cu e danos ao DNA nas brânquias dos peixes, onde estes efeitos são recuperados após 240 h em água do mar sem adição de Cu.

Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress.

The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.

Hepatocyte apoptosis in dairy cattle during the transition period.

The objective of this study was to investigate hepatocyte apoptosis in dairy cows during the transition period. Four clinically healthy, pregnant dairy cattle were used. The cows had no clinical diseases throughout this study. Blood samples were collected and livers were biopsied from the cows at 3 different times: 3 weeks before expected partition (wk -3); during parturition (wk 0), and 3 weeks (wk +3) after parturition. The damage to deoxyribonucleic acid (DNA) caused by hepatocytes was evaluated by comet assay. The apoptotic features of hepatocytes were examined by immunohistochemistry and electron microscopic analyses. The hepatic triglyceride content markedly increased at wk 0 and wk +3 compared with the values at wk -3. The results of the comet assay showed increases in the mean tail moment values of hepatic cells after parturition in all cows, which suggested increased DNA damage. Histopathologically, the hepatocytes began to contain lipid droplets at wk 0 and were severely opacified at wk +3. Caspase-3-positive and single-stranded DNA-(ssDNA)-positive cells were first detected in the liver after parturition. Condensation of nuclear chromatin, a typical sign of apoptosis, was confirmed by transmission electron microscopy after parturition. These results suggest that apoptosis is induced in hepatocytes of dairy cows around parturition and may result from lipotoxicity in hepatocytes.

L'objectif de ce travail était d'étudier l'apoptose des hépatocytes chez les vaches laitières durant la période de transition. Quatre vaches laitières gestantes et cliniquement saines faisaient partie de l'étude. Les vaches n'ont présenté aucune maladie clinique tout au long de l'étude. Des échantillons sanguins ont été prélevés et des biopsies hépatiques obtenues à partir des animaux à trois moments différents : 3 semaines avant la date prévue de parturition (sem −3); durant la parturition (sem 0) et 3 semaines après la parturition (sem +3). Les dommages à l'ADN causés par les hépatocytes ont été évalués par l'essai Comet. Les caractéristiques apoptotiques des hépatocytes ont été examinées par analyses immunohistochimiques et par microscopie électronique. Le contenu hépatique en triglycéride augmenta de manière marquée aux sem 0 et +3 comparativement à la valeur observée à sem −3. Les résultats de l'essai Comet ont montré pour les hépatocytes de toutes les vaches après parturition des augmentations des valeurs du moment moyen de la queue, ce qui suggérait une augmentation des dommages à l'ADN. À l'examen histopathologique, les hépatocytes commencèrent à contenir des gouttelettes lipidiques à la sem 0 et étaient sévèrement opacifiés à la sem +3. Les cellules positives pour la caspase-3 et celles positives pour de l'ADN simple brin ont été les premières à être détectées dans le foie après la parturition. La condensation de la chromatine nucléaire, un signe typique d'apoptose, a été confirmée par microscopie électronique à transmission après la parturition. Ces résultats suggèrent que l'apoptose est induite dans les hépatocytes des vaches laitières aux alentours de la parturition et pourrait résulter d'une lipotoxicité dans les hépatocytes.(Traduit par Docteur Serge Messier).

Increased DNA damage and oxidative stress in chickens with natural Marek's disease.

Oxidative stress contributes to the accumulation of genomic abnormalities, prevents cellular apoptosis, and also mediates immunosuppression resulting in tumor formation. Marek's Disease provides excellent opportunities for the study of herpesvirus-induced tumors both in experimental- and natural conditions. The aim of this study was to examine the effects of Marek's Disease (MD) on basal levels of DNA strand breaks and on the oxidative-antioxidative status of chickens with MD. White-Lohmann hens-fifteen infected with Marek's Disease Virus (MDV) and fifteen healthy-of same age and sex were included in this study. MD infection was diagnosed via clinical signs, gross- and micro-pathological findings and also by detection of viral antigens in feather follicle epithelium by the indirect immunoperoxidase method. Compared with healthy controls, DNA damage was greater and levels of malondialdehyde (MDA) and plasma protein carbonyl (PCO), and plasma concentration of nitric oxide metabolites (NOx) higher in the MD group. Furthermore, total antioxidant activities (AOAs) were found lowered and glutathione (GSH) levels reduced in the MD group compared to the control group. Significantly positive correlation was found between DNA damage, MDA, PCO, and NOx in the MD group. DNA strand breaks were found negatively associated with AOA and GSH concentrations in the MD group. Our results demonstrated that oxidative stress markers and DNA damage substantially increased in chickens with MD, which indicated that increased DNA damage levels might be related to the increased oxidative stress and reduced antioxidant activity.

Single-dose pharmacokinetics and genotoxicity of metronidazole in cats.

Single-dose pharmacokinetics and genotoxicity of metronidazole in cats were evaluated. Cats received either 5mg/kg metronidazole intravenously, or 20mg/kg metronidazole benzoate (12.4mg/kg metronidazole base) orally in a single dose. Serial plasma samples were collected and assayed for metronidazole using high pressure liquid chromatography (HPLC). Genotoxicity was assessed in vitro in feline peripheral blood mononuclear cells (PBMC) and a feline T-cell lymphoma line incubated with metronidazole, and in vivo in PBMC collected before, during and 7 days after oral metronidazole, by use of the COMET assay. Systemic absorption of metronidazole was variable (mean=65+/-28%) with a peak of 8.84+/-5.4microg/ml at 3.6+/-2.9h. The terminal half-life was 5.34h from the intravenous dose and 5.16h from the oral dose. Systemic clearance was low (mean=91.57ml/h/kg [1.53ml/kg/min]), and the apparent volume of distribution (steady state) was 0.650+/-0.254l/kg. Genotoxicity was detected at all concentrations of metronidazole in feline PBMC and the T-cell lymphoma line in vitro. Genotoxicity was also observed in PBMC collected from cats after 7 days of oral metronidazole but resolved within 6 days of discontinuing metronidazole.

Mutation analysis of ras gene in the liver of European eel (Anguilla anguilla L.) exposed to benzo[a]pyrene.

Ras is regarded as one of the most important genes involved in carcinogenesis. Such genes have been characterised in several fish species and the presence of ras mutations have already been described in fish populations from hydrocarbon contaminated areas and following experimental exposure to specific contaminants. The aims of this study were to evaluate the DNA integrity by comet assay, to isolate the normal ras gene of Anguilla anguilla and analyse for the presence of ras gene mutations or changes in gene expression levels induced after one month of benzo[a]pyrene (BaP) experimental exposure. The A. anguilla ras cDNA isolated revealed a 189 amino acid protein and alignment with other vertebrate ras proteins revealed conservation of functionally important regions. Following experimental exposure to BaP, an increase in DNA damage was found by comet assay. However, no point mutations or changes in ras gene expression levels were detected when compared to control samples. In contrast to the majority of fish ras gene sequences, a high degree of polymorphic variation was detected in the A. anguilla ras gene.

Evaluation of relationships between reproductive metrics, gender and vitellogenin expression in demersal flatfish collected near the municipal wastewater outfall of Orange County, California, USA.

Estrogenic activity in fish has primarily been evaluated using vitellogenin (vtg) expression in male and juvenile animals. Although the response has been widespread in field and laboratory studies, the relevance of the response to higher level adverse effects, particularly in the field, is less than clear. Previous evaluations of vtg within flatfish species collected near the Orange County Sanitation District (OCSD) outfall and stations as far as 7.7 km down current indicated bioavailable estrogens within demersal flatfish populations. In order to evaluate the persistence of estrogenic activity and relationships to reproduction and development, fish were sampled in the winter and summer of 2003 and 2004 at the outfall and a reference location. Vtg, plasma estradiol (E2) concentrations, gonadosomatic indices (GSI), sperm DNA damage, development, and gender ratios were measured in English Sole (Pleuronectes vetulus) and Hornyhead Turbot (Pleuronichthys verticalis). Variable levels of vtg were continually observed in the plasma samples of fish collected at both locations. Vtg expression and plasma E2 levels were significantly correlated in females. A positive relationship was demonstrated between plasma E2 levels and sperm DNA damage. Rather than an expected feminization of populations, a trend toward masculinization was observed particularly at the OCSD outfall, as indicated by gender ratios and significantly higher GSI in males versus females. These results are consistent with previous studies showing vtg expression in male flatfish, but no alteration in overall flatfish abundance at the sampled sites.

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

Bull spermatozoa under mercury stress.

Defective sperm function is the most prevalent cause of male infertility and is difficult to treat. This study was designed to evaluate the effect of mercuric chloride (HgCl2) at 50-300 micromol/l concentration range, in vitro, on the sperm membrane and DNA integrity, viability, reduced glutathione (GSH) content and acrosomal status of the bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid [phosphate-buffered saline (PBS), pH 7.2]. I recorded a meaningful increase in the lipid peroxidation (LPO) rate and a drastic fall in the spermatocrit values under mercury additions, dominantly at 300 microM mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage of viable spermatozoa (r = -0.9, p < 0.001). GSH content was significantly impaired. Data obtained from Comet assay [single-cell gel electrophoresis (SCGE)] technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 90% of DNA breaks were double-stranded. The correlation between LPO rate and percentage of DNA breaks was found to be 0.9 (p < 0.001). Results of the gelatin test indicate that mercury is capable of altering the integrity of acrosomal membranes, showing an abnormal acrosome reaction. In this regard, a strong correlation was found between LPO rate and percentage of halos (r = -0.9, p < 0.001). In conclusion, mercury proved to be a potential oxidant in the category of 'environmental factors' to bull spermatozoa. Hence, considering the widespread use of mercury and its compounds, these metals should be regarded with more concern.

The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay.

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by gamma-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells.