'Site of contact genotoxicity' assessment for implants - Potential use of single cell gel electrophoresis in biocompatibility testing of medical devices.

Toxicological risk assessment of medical devices requires genotoxicity assessment as per ISO 10993, Part 3, which is designed to address gene mutations, clastogenicity and/or aneugenicity endpoints. 'Site of contact genotoxicity' is a potential genotoxic risk especially for medical implants, that is currently not addressed in biocompatibility standards. We therefore performed initial validation study on the use of alkaline single cell gel electrophoresis (comet assay) for detecting 'site of contact genotoxicity' of medical devices, using test items made of acrylic implants impregnated with ethyl methanesulphonate (EMS). Comet assay detected increased DNA migration at the site of implantation, but not in the liver. The same implants also failed to show any genotoxicity potentials, when tested on the standard test battery using Salmonella/microsome and chromosome aberration assays. The study suggested that some medical implants can cause 'site of contact genotoxicity', without producing systemic genotoxicity. In conclusion, comet assay will add new dimension to safety assessment of medical devices, and this assay can be added to the battery of genetic toxicology tests for evaluating biocompatibility of medical implants.

Dosage time affects alkylating agents induced micronuclei in mouse peripheral blood reticulocytes through the function of erythropoietin.

Previously, we reported that the frequency of micronucleated reticulocytes (MNRETs) in the peripheral blood of male C3H/He mice intraperitoneally administered ethylnitrosourea (ENU) (25 mg/kg body weight) in the dark period (zeitgeber time, ZT15) was higher than in the light period (ZT3). In this study, to clarify the mechanism underlying this phenomenon, we investigated the differences in micronucleus (MN) induction observed between ZT3 and ZT15 using five chemicals, methylnitrosourea (MNU), ethylmethane sulfonate (EMS), mitomycin C, cyclophosphamide and vincristin. MNU and EMS, monofunctional alkylating agents, showed higher frequencies of MNRETs in the ZT15 than the ZT3 treatment similar to ENU. However, no differences were observed for the other chemicals. In the comet assay, more DNA damage was induced by ENU in the ZT15 than the ZT3 treatment. Furthermore, the plasma erythropoietin (EPO) level, a known effector of MN induction with anti-apoptotic activity mediated by Bcl-xL expression, was higher in the dark than in the light period. EPO did not increase the frequency of MNRETs. However, in the ENU treatment group at ZT3 following EPO injection a significant increase of MNRETs was observed similar to the ZT15 treatment. Higher expression of apoptosis-related genes such as Bcl-xL was induced in bone marrow cells from mice treated with ENU at ZT15 compared with ZT3. From these results, it was speculated that the differences in MN induction in the peripheral blood of mice exposed to monofunctional alkylating agents such as ENU depend on apoptotic or anti-apoptotic conditions related to the circadian rhythms of EPO in bone marrow.

Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate.

Ser139-phosphorylated H2AX (γH2AX) is a functional biomarker of DNA double-strand breaks. However, its conventional detection for in vivo samples relies on immunological methods using anti-γH2AX antibodies, making quantitative analysis difficult. Here, we established an absolute γH2AX quantification in vivo method for multiple organs in mice using liquid chromatography-triple quadrupole tandem mass spectrometry. When applying the method to male Institute of Cancer Research (ICR) mice (8 weeks old), the testes showed the highest γH2AX level (2.3% of total H2AX), followed by the bone marrow (0.51%), stomach (0.28%), kidney (0.20%), spleen (0.20%), liver (0.15%) and lung, which had the lowest overall level (0.10%). After intraperitoneal administration of 2 mg/kg mitomycin C in mice, γH2AX levels increased until 2-4 h, followed by a monotonical decrease to the control level in the bone marrow and spleen, and increased moderately until 24 h, followed by a slight decrease by 48 h in the liver, stomach, lung and kidney. After oral administration of 400 mg/kg ethyl methanesulphonate, γH2AX levels increased until 8 h and then decreased to the control level by 24-48 h in the spleen and kidney, increased until 24 h and then slightly decreased until 48 h in the bone marrow and lung, increased until 8 h and plateaued by 48 h in the liver, and decreased until 8 h and then increased to the control level in the stomach. Both the genotoxic chemicals did not alter γH2AX levels in the testes. These results indicate that our novel method could reveal variation in the γH2AX state in mouse organs and allows monitoring of the in vivo dynamics induced by genotoxic chemicals.

Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes.

In cardiac myocytes, an enhancement of late sodium current (INaL) under pathological conditions is known to cause prolongation of action potential duration (APD). This study investigated the contribution of INaL under basal, physiological conditions to the APD Whole-cell INaL and the APD of ventricular myocytes isolated from healthy adult guinea pigs were measured at 36°C. The INaL inhibitor GS967 or TTX was applied to block INaL The amplitude of basal INaL and the APD at 50% repolarization in myocytes stimulated at a frequency of 0.17 Hz were -0.24 ± 0.02 pA/pF and 229 ± 6 msec, respectively. GS967 (0.01-1 µmol/L) concentration dependently reduced the basal INaL by 18 ± 3-82 ± 4%. At the same concentrations, GS967 shortened the APD by 9 ± 2 to 25 ± 1%. Similarly, TTX at 0.1-10 µmol/L decreased the basal INaL by 13 ± 1-94 ± 1% and APD by 8 ± 1-31 ± 2%. There was a close correlation (R2 = 0.958) between the percentage inhibition of INaL and the percentage shortening of APD caused by either GS967 or TTX MTSEA (methanethiosulfonate ethylammonium, 2 mmol/L), a NaV1.5 channel blocker, reduced the INaL by 90 ± 5%, suggesting that the NaV1.5 channel isoform is the major contributor to the basal INaL KN-93 (10 µmol/L) and AIP (2 µmol/L), blockers of CaMKII, moderately reduced the basal INaL Thus, this study provides strong evidence that basal endogenous INaL is a significant contributor to the APD of cardiac myocytes. In addition, the basal INaL of guinea pig ventricular myocytes is mainly generated from NaV1.5 channel isoform and is regulated by CaMKII.

In-utero exposure to nelfinavir-ethyl methyl sulfone.

Ethyl methyl sulfone contained in nelfinavir between 2007 and 2008 accidentally exposed embryos and fetuses to a powerful mutagen. We report data for 101 HIV-uninfected children exposed in utero included in the French prospective national cohort. The incidence of malformation was similar to that in the cohort as a whole with different drug exposures; no children had developed cancer after 9 years of follow-up.

Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

Utility of RAPD marker for genetic diversity analysis in gamma rays and ethyl methane sulphonate (EMS)-treated Jatropha curcas plants.

The presence of important chemical and physical properties in Jatropha curcas makes it a valuable raw material for numerous industrial applications, including the production of biofuel. Hence, the researcher's interest is diversified to develop more and better varieties with outstanding agronomic characteristics using conventional breeding. Among these, mutation breeding is one of the best approaches to bring genetic changes in plant species. The aim of this study is to evaluate the diversity and genetic relationship among J. curcas mutants, which were obtained from different doses of gamma rays (control, 5 Kr, 10 Kr, 15 Kr, 20 Kr and 25 Kr) and EMS (1%, 2%, 3% and 4%), using RAPD marker. Among the 21 random primers, 20 produced polymorphic bands. The primers, OPM-14 and OPAW-13, produced a minimum number of bands (3) each across the ten mutants, while the primer OPF-13 produced the maximum number of bands (10), followed by the primers OPU-13, OPAM-06, OPAW-09 and OPD-05, which produced 9 bands each. The number of amplicons varied from 3 to 10, with an average of 7 bands, out of which 4.57 were polymorphic. The percentage of polymorphism ranged from 0.00 to 100 with an average of 57%. In the present study, RAPD markers were found most polymorphic, with an average polymorphism information content (PIC) value of 0.347, effective multiplex ratio (EMR) of 35.14, marker index (MI) of 14.19, resolution power (Rp) of 11.19, effective marker index (EMI) of 8.21 and genotype index (GI) of 0.36, indicating that random primers are useful in studies of genetic characterization in J. curcas mutant plants. In a dendrogram constructed based on Jaccard's similarity coefficients, the mutants were grouped into three main clusters viz., (a) control, 10 Kr, 15 Kr, 20 Kr, 2% EMS, and 3% EMS, (b) 5 Kr and 1% EMS, and (c) 25 Kr and 4% EMS mutants. Based on the attributes of the random primers and polymorphism studied, it is concluded that RAPD analysis offers a useful molecular marker for the identification of the mutants in gamma rays and EMS treated plants.

The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells.

The X-linked PIG-A gene is involved in the biosynthesis of the cell surface anchor GPI, and its inactivation may serve as a new marker for mutagenicity. The in vivo PIG-A gene mutation assay is currently being validated by several groups. In this study, we established a corresponding in vitro variant of the PIG-A assay applying B-lymphoblastoid TK6 cells. PE-conjugated antibodies against the GPI-anchored proteins CD55 and CD59 were used to determine the GPI status via multicolor flow cytometry. Mutant spiked TK6 cell samples were analyzed, and mutants were quantified with even small numbers being quantitatively recovered. To validate our approach, mutant spiked cell samples were analyzed by flow cytometry and proaerolysin selection in parallel, yielding a high correlation. Further, we developed a procedure to reduce the background level of preexisting mutant cells to lower than 20 in 10(6) cells to increase the sensitivity of the assay. Spontaneous rate of GPI deficiency was investigated being 0.76 × 10(-6)/cell/generation for TK6 cells. The optimal phenotype expression time after ethyl methanesulfonate treatment was found to be 10 days. We applied the in vitro PIG-A assay to demonstrate the mutagenicity of ethyl methanesulfonate, 4-nitroquinoline 1-oxide and UV-C irradiation in a dose-dependent and statistically significant manner. Pyridine and cycloheximide were included as negative controls providing negative test results up to 10 mM. These data suggest that the in vitro PIG-A assay could complement the in vivo PIG-A assay with some distinct advantages compared to other in vitro mammalian mutagenicity tests.

A new system for detecting mutations in arabidopsis thaliana and the mutational spectra resulting from ethylmethanesulfonate treatment.

A system was developed for the detection and analysis of mutations occurring on chromosomal DNA in plants. The plasmid pML4, carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector, pCGN5138, to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plants exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to isolate mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E. coli cells resistant to streptomycin. Here we report that the system could detect the mutagenic effect of ethylmethanesulfonate (EMS). Further characterization of the mutants revealed that G:C to A:T transitions predominated among the EMS-induced mutations. This assay system is useful for the detection and analysis of mutations arising on chromosomal DNA in plants, and should be useful for evaluating analysis of the effects of environmental mutagens.

[Biological effect of low doses of alkylating agents in drosophila studies]. / Vliianie malykh doz na biologicheskie éffekty alkiliruiushchikh agentov v issledovaniiakh na drozofile.

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.

Anti-genotoxicity of trans-anethole and eugenol in mice.

The naturally occurring flavouring agents trans-anethole and eugenol were evaluated for antigenotoxic effects in mice. The test doses of trans-anethole (40-400 mg/kg body weight) and eugenol (50-500 mg/kg weight) were administered by gavage 2 and 20 h before the genotoxins were injected intraperitoneally. Anti-genotoxic effects were assessed in the mouse bone marrow micronucleus test. Pretreatment with trans-anethole and eugenol led to significant antigenotoxic effects against cyclophosphamide (CPH), procarbazine (PCB), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and urethane (URE). In addition, trans-anethole inhibited the genotoxicity of ethyl methane sulfonate (EMS). Both trans-anethole and eugenol exerted dose-related antigenotoxic effects against PCB and URE. There was no significant increase in genotoxicity when trans-anethole (40-400 mg/kg body weight) and eugenol (50-500 mg/kg body weight) were administered alone.

Inducible protective processes in animal systems VI. Cross-adaptation and the influence of caffeine on the adaptive response in bone marrow cells of mouse.

The effect of caffeine (CAF) (a replicative DNA synthesis inhibitor) given as pre-, inter- and post-treatments on the ethyl methanesulfonate (EMS)-induced adaptive response in in vivo mouse bone marrow cells was studied in order to understand the influence of CAF on the adaptive response. The pre-treatment was given 4 h before a combined treatment with EMS (conditioning + challenge) and in another set CAF was given as a conditioning dose and 4 h later the cells were challenged with a high dose of EMS. In the inter-treatment, CAF (40 mg/kg body wt) was administered 2 or 4 h after the conditioning dose of EMS and 6 or 4 h later the cells were challenged with a high dose of EMS. Similarly, in the post-treatment experiments, CAF was injected 6, 12 or 18 h after a combined treatment with EMS. The results revealed that the pre-, inter- and post-treatments with CAF significantly reduced the frequency of chromosomal aberrations compared with the challenge and combined treatments with EMS. It is interesting to note that CAF pre-treatment resulted in a much greater reduction in chromosomal aberrations compared with the inter- and post-treatments. Thus, this is an example of cross-adaptation induced by CAF in EMS-treated in vivo mouse bone marrow cells and the results also demonstrate an influence of CAF on the adaptive response.

Identification of the mgc1 mutation which affects mating-pheromone-induced morphogenesis in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae undergo morphogenesis in response to the mating pheromones a- and alpha-factors. The 'shmoo' morphology involves localized cell surface projection formation and cytoskeleton protein synthesis. This polarization is presumed to be a prelude to mating between mating partners with opposite mating types, a and alpha. To identify genes involved in pheromone-induced morphogenesis, a system in which a gpa1 fus3 double mutant was used as a parent strain was developed, and mutants which showed altered morphogenesis in response to mating pheromone were identified. The mutation was designated mgc1 for morphogenesis control by mating pheromones. The mgc1 mutant arrested cell division in response to alpha-factor and mated with cells of the opposite mating type, but did not form a typical projection in response to pheromones.

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes.

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.

Micronucleus test with ethyl methanesulfonate administered by intraperitoneal injection and oral gavage.

The effect of route of administration on the outcome of the micronucleus test was studied by administering ethyl methanesulfonate (EMS) by oral gavage (p.o.) and intraperitoneal injection (i.p.) to males of 2 mouse strains, MS/Ae and CD-1. Based on preliminary studies, consisting of a small-scale acute toxicity test and a pilot experiment to determine the optimal sampling time and the appropriate dosages, a micronucleus test was conducted with a 24-h sampling time and doses of 50-400 mg/kg i.p. and p.o. EMS significantly induced micronucleated polychromatic erythrocytes (MNPCEs) with a clear positive dose response by both routes in both strains. Moreover, both routes showed almost the same induction rate of MNPCEs at each dose level tested in both strains.

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells.

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.

Efficiency of different methods of EMS application for the induction of dominant lethals in germ cells of medfly, Ceratitis capitata (Wied.), males.

EMS (ethyl methanesulfonate) fed to adult Mediterranean fruit flies in 10% sugar water was found to be the most effective treatment for the induction of dominant lethals in male germ cells. This application procedure showed a direct regression between the log concentration of EMS and the probit F1 egg lethality, provided a reasonably uniform uptake of EMS by the exposed males, and was non-toxic at the relevant concentrations. The same application procedure, but employing 1% sugar water, was also non-toxic to the treated males but resulted in large variations in the rate of uptake of the mutagen, thus producing no clear correlation between the concentration of EMS and dominant lethality. Injection of adult males with EMS caused high parental mortality and caused a severe reduction in mating propensity at concentrations below that causing dominant lethality. Dominant lethality was observed in all treatment procedures as a reduction in egg hatchability, whereas adult emergence from surviving pupae was never affected. A small, but significant, reduction in pupal production from hatched eggs was observed in the treatment involving "egg/larval feeding" and in all adult treatments, but in no case could this be correlated to the concentration of EMS. The high levels of radioactivity, observed in the testes of males treated with 14C-labelled EMS through feeding of adults (10% sugar), in spermathecae of females mated to these males and in resultant F1 eggs, suggest that a major portion of the label reaching the testes was associated with the sperm itself rather than with other parts of the testes or the seminal fluid.

The role of feeding rejection in Drosophila mutation assays.

The frequencies of sex-linked recessive lethal mutations recovered in the male post-meiotic germ cells of Drosophila after feeding on solutions containing a mutagen (either 1 mM methyl methanesulfonate, MMS or 2 mM ethyl methanesulfonate, EMS) and 5-bromo-2-deoxyuridine (BUdR) were significantly lower than the frequencies observed after treatment with the mutagen alone. In an attempt to explain the apparent 'protective' effect of BUdR, the feeding behavior of the flies was monitored for differences in the uptake of the mutagen-containing solution in the presence and absence of BUdR. This was accomplished by measuring the uptake of [14C]sucrose. The results indicated that the uptake of the feeding solution is inhibited by the presence of the selected concentration of BUdR (1.0 or 32.5 mM). Such a reduced uptake of the mutagen could alone account for the reduction in mutational yields noticed in treatments containing mutagen + BUdR compared to the ones with the mutagen alone. These results emphasize the need to monitor the feeding behavior of flies in experiments involving adult feeding.