Clastogenic and Mutagenic Actions of Active Species Generated in the 6-Hydroxydopamine/Oxygen Reaction: Effects of Scavengers of Active Oxygen, Iron, and Metal Chelating Agents

A pro-oxidant triphenol, 6-hydroxydopamine (6-OHDA), induced mutations in the Salmonella typhimurium TA104 tester strain (over the concentration range to 800/JM), and induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells at lower concentrations (up to 90 μM). It was however only marginally mutagenic (up to cytotoxic levels of 200 μM) in the TA102 tester strain. Clastogenicity in the more sensitive CHO cell assay was mediated by activated oxygen. Superoxide dismutase decreased the incidence of chromosomal aberrations by 60% and catalase (or superoxide dismutase plus catalase) decreased the incidence to control levels. The clastogenicity of 6-OHDA was dependent upon unsequestered transition metal ions, since addition of EDTA plus desferoxamine decreased chromosomal aberrations by 90%. The simplest explanation of the data is that genotoxicity is mediated by active species generated in a Fenton-type reaction between 6-OHDA and H₂O₂ catalyzed by traces of metals in the medium.     

Chemotherapeutic drugs induced female reproductive toxicity and treatment strategies

Increase in success of cancer treatment with advancement in the screening, prognosis and diagnosis protocols have significantly improved the rate of cancer survivorship. With the declining cancer mortality, however, the cancer survivors are also subjected to the adverse consequences of chemotherapy, particularly in the female reproductive system. Recent studies have shown the sensitivity of the ovarian tissue to the chemotherapeutic drugs-induced toxicity. Several in vitro and in vivo studies have assessed the toxic effects of chemotherapeutic drugs. The most frequently used chemotherapeutic drugs such as doxorubicin, cyclophosphamide, cisplatin and paclitaxel have been reported to cause ovarian damage, diminution of follicular pool reserve, premature ovarian failure and early menopause, resulting into declining fertility potential among females. The chemotherapy often employs combination of drug regimen to increase the efficacy of the treatment. However, the literature mostly consists of clinical data regarding the gonadotoxicity caused by anticancer drugs but there lacks the understanding of toxicity mechanism. Therefore, understanding of the different toxicity mechanisms will be helpful in development of possible therapeutic interventions for preservation of declining female fertility among cancer survivors. The current review comprehends the underlying mechanisms of female reproductive toxicity induced by the most commonly used chemotherapeutic drugs. In addition, the review also summarizes the recent findings related to the use of various protectants to diminish or at least in managing the toxicity induced by different chemotherapeutic drugs in females.     

DNA-damage induction by eight metal compounds in TK6 human lymphoblastoid cells: Results obtained with the alkaline Comet assay

Metal compounds are long-lived and can react with different macromolecules, producing a wide range of biological effects, including DNA damage. Since their reactivity is associated with their chemical structure, it is important to obtain information on more than one compound from the same metal. In this study, the DNA-damaging potential of two mercury compounds (mercury chloride and methyl mercury chloride), two nickel compounds (nickel chloride and potassium hexafluoronickelate), two palladium compounds (ammonium tetrachloropalladate and ammonium hexachloropalladate), and two tellurium compounds (sodium tellurite and sodium tellurate) was evaluated in human lymphoblastoid TK6 cells by use of the alkaline version of the Comet assay. As the use of computerized image-analysis systems to collect comet data has increased, the metric used for quantifying DNA damage was the Olive tail moment. Treatments lasted for 3 h and the range of concentrations tested was different for each metal compound, depending on its toxicity. Both mercury agents produced DNA damage in TK6 cells, with mercury chloride producing considerably more DNA damage than methyl mercury chloride. Of the two nickel compounds, only nickel chloride (a Ni(II) compound) induced DNA breaks. Similarly, of the two palladium compounds, only the Pd(II) compound (ammonium tetrachloropalladate) was positive in the assay. Sodium tellurite was clearly positive, producing concentration-related increases in DNA damage, while sodium tellurate gave a negative response. In conclusion, the ability of inducing DNA damage by the selected metal compounds in human TK6 cells, when measured with the Comet assay, was dependent on the chemical form and, in general, compounds containing the metal in the lower valence state displayed the greater DNA-damaging ability.     

Extracts affecting mitotic division in root-tip meristematic cells

Some species of Psychotria (Rubiaceae) are important in herbal medicine, where their extracts are used internally for infections of the female reproductive system, bronchitis, gastrointestinal disturbances, skin irritations, tumors, ulcers, and eye disturbances. The antiproliferative and genotoxic effects of Psychotria myriantha Mull. Arg. and P. leiocarpa Cham. et Schlecht infusions on the Allium cepa L. cell cycle were evaluated. The teas were prepared by infusing the leaves in distilled water, in two concentrations: 1.13 mg/mL and 6.78 mg/mL. Three groups of four bulbs were used for each Psychotria species. After the bulbs rooted in distilled water, they were transferred to the teas for 24 hours, except for the control that stayed in water. The rootlets were then collected, fixed in ethanol-acetic acid (3:1) for 24 hours, and stored in 70% ethanol. For each group of bulbs, 4000 cells were analyzed, calculating the mitotic indexes, submitting them to statistic analysis, using the χ ² test (p = 0.05). The results showed a decrease in mitotic index with an increase in tea concentration in both species. In P. leiocarpa, the mitotic index values differed significantly between the control and concentration of 6.78 mg/mL (χ ² = 9.863). For P. myriantha, the values referring to the mitotic index differed greatly between the control and the treatments (χ ² = 124.8). With this study, it was determined that P. myriantha and P. leiocarpa infusions possess antiproliferative effects on the A. cepa cell cycle, and teas of P. myriantha also have genotoxic activity.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

In vitro genotoxicity of terbutryn evaluated by the alkaline single-cell microgel-electrophoresis "comet" assay

Terbutryn, as-triazine herbicide, is extensively used in agriculture as a selective pre- and postemergence control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Terbutryn was reported to degrade slowly, with half-lives of 240 and 180 days in pond and river sediment, respectively. The tendency of this herbicide to move from treated soils to water compartments through water runoff and leaching was demonstrated and residual amounts of terbutryn and its metabolites have been found in drinking water, and industrial food products, long after application. Although this herbicide may be regarded as a contaminant of our environment, only limited and inconsistent data exist concerning its genotoxic properties. In this study, the DNA-damaging ability of the herbicide was evaluated in the alkaline single-cell microgel-electrophoresis ("comet") assay by testing terbutryn in the presence of S9mix (rat liver homogenate containing microsomal enzymes plus cofactors) prepared with liver homogenate from both uninduced (basal) and aroclor 1254-induced rats. DNA damage was recorded in freshly isolated human peripheral blood leukocytes. A statistically significant increase in the extent of primary DNA damage, more pronounced in the absence of S9mix, took place only when terbutryn concentrations were high (100 and 150 μg/ml), in the presence of a concomitant mild cytotoxic effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotoxic and Mutagenic Assessment of Hexavalent Chromium in Fish Following In Vivo Chronic Exposure

Chromium is a well-documented carcinogen. To evaluate the genotoxic potential of hexavalent chromium on an aquatic bio-system, freshwater murrel fish (Channa punctatus) were exposed to potassium dichromate. The 96-h LC₅₀ for potassium dichromate was 61.80 mg/L for the test fish in a static system. On the basis of the 96-h LC₅₀, fish were exposed to sublethal concentrations of the test chemical. Fish exposed to the test chemical were sampled on days 1, 7, 14, 21, and 28 post-exposure and blood and gill cells were collected. Significantly (p < .05) higher DNA damage in both lymphocyte and gillcells and micronuclei formation in whole blood was observed at different test concentrations and sampling times of the test chemical as compared to control fish. The mean% tail DNA in the comet tail assay showed a concentration-dependent increase and the maximum% tail DNA was observed on day 7 of exposure in both cells. A similar trend was also observed in micronuclei induction in blood with maximum induction on day 21. Hexavalent chromium showed genotoxic potential in chronic exposure of C. punctatus, and the micronucleus test and the comet assay are the methods for sensitive and rapid detection of the genetic effects.     

In vitro genotoxic effects of titanium dioxide nanoparticles (n‐TiO2) in human sperm cells

Titanium dioxide nanoparticles (TiO₂‐NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO ₂‐NPs genotoxic effects on human spermatozoa in vitro. TiO ₂‐NPs are able to cross the blood–testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n‐TiO ₂ concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n‐TiO ₂ genotoxicity. Primary n‐TiO ₂ characterization was performed by transmission electron microscopy. The dispersed state of the n‐TiO ₂ in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD‐PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD‐PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n‐TiO ₂. The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n‐TiO ₂ exposure. This research provides the evaluation of n‐TiO ₂ potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.     

Assessment of titanium dioxide nanoparticles toxicity via oral exposure in mice: effect of dose and particle size

Objective: The aim of the present work is to evaluate the toxicity of titanium dioxide nanoparticles (TiO₂NPs) according to their doses and particle sizes.

Materials and methods: The effect of five days oral administration of TiO₂NPs (21 and 80?nm) with different doses (50, 250 and 500?mg/kg body weight) was assessed in mice via measurement of oxidative stress markers; glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and nitric oxide (NO), liver function indices; aspartate and alanine aminotransferases (AST and ALT), chromosomal aberrations and liver histopathological pattern.

Results: The results revealed drastic alterations in all the measured parameters and showed positive correlation with the gradual dose increment. In addition, the smaller particle size of TiO₂NPS (21?nm) had more adverse effect in all the selected biochemical parameters, genetic aberrations and histological investigations.

Conclusions: Toxicity of TiO₂NPs increases in a dose-dependent manner and vice versa with particles size. The evaluated biomarkers are good indicators for TiO₂NPs toxicity. More detailed studies are required before the recommendation of TiO₂NP_S as food additives.     

Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes

Several studies have demonstrated beneficial effects of supplemental trivalent Cr in subjects with reduced insulin sensitivity with no documented signs of toxicity. However, recent studies have questioned the safety of supplemental trivalent Cr complexes. The objective of this study was to evaluate the cytotoxic and genotoxic potential of the Cr(III) complexes (histidinate, picolinate, and chloride) used as nutrient supplements compared with Cr(VI) dichromate. The cytotoxic and genotoxic effects of the Cr complexes were assessed in human HaCaT keratinocytes. The concentrations of Cr required to decrease cell viability were assessed by determining the ability of a keratinocyte cell line (HaCaT) to reduce tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. DNA damage using the Comet assay and the production of 8-hydroxy-2′-deoxyguanosine were also determined with and without hydrogen peroxide-induced stress. The LC50 for human cultured HaCaT keratinocytes was 50 μM for hexavalent sodium dichromate and more than 120-fold higher for Cr chloride (6 mM) and Cr histidinate (10 mM). For Cr picolinate at saturating concentration (120 μM) the LC50 was not attained. High Cr(III) concentrations, 250 μM Cr as Cr chloride and Cr histidinate and 120 μM Cr picolinate (highest amount soluble in the system), not only did not result in oxidative DNA damage but exhibited protective antioxidant effects when cells were exposed to hydrogen peroxide-induced oxidative stress. These data further support the low toxicity of trivalent Cr complexes used in nutrient supplements.