Vincent Ferrier

In vivo detection of waste water and industrial effluent genotoxicity: use of the Newt Micronucleus test (Jaylet test)

The genotoxic potential of various waste waters has been evaluated in a micronucleus test using amphibian larvae. Genotoxicity was detected after dilution, in waste water from tanneries and from various petrochemical industries. Further studies have shown that sample treatment used for in vitro testing may affect the genotoxic response. Sterilization by gamma irradiation lowered genotoxic activity. Furthermore, microfiltration of effluent and extraction of organic micropollutants on XAD-4 resins, lead to the preparation of extracts which are not fully representative of the initial water sample. Testing of concentrates, as required for in vitro studies, will limit the scope of a survey to that part of the organic matter that can be recovered by concentration techniques. Many of the problems encountered in in vitro genotoxicity studies of waters, may be circumvented with direct testing on aquatic organisms. Thus, there is no need to concentrate or sterilise a sample. The tests can be carried out with intact animals, thus taking into account uptake and elimination, internal transport and metabolism. Finally, in vivo test-systems, such as the Newt Micronucleus Test, are more relevant to eukaryotes than bacterial assays and are suitable to assess the real impact of genotoxins discharged in the aquatic environment.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele Amphibian). II. Peptidases: Demonstration of sex linkage

The existence of four peptidases was demonstrated by starch gel electrophoresis in Pleurodeles waltlii: PEP-1, PEP-2, PEP-3, and PEP-4. Peptidases-3 and -4 are monomorphic, and peptidases-1 and -2 are polymorphic. The heredity of the polymorphisms was studied using individuals arising from crosses or of gynogenetic origin. Peptidase-1 is dimeric; its polymorphism depends on a pair of codominant alleles, Pep-1A and Pep-1B, which are situated on the Z and W sex chromosomes, respectively, in close proximity to, or even within, the sex differential segment. As the differential segment is very close to the centromere, the PEP-1 locus therefore also appears to be closely linked to it. Expression of the PEP-1 locus was shown to be independent of the sex hormone environment. This locus is the first case reported in amphibians of an enzyme marker linked to the genetic sex. It allows the sex of Pleurodeles to be determined before they reach sexual maturity. Peptidase-2 is monomeric. Its polymorphism depends on a pair of codominant alleles on an autosomal PEP-2 locus. The high proportion of heterozygous animals in the gynogenetic offspring of females heterozygous for the PEP-2 locus indicates segregation which is independent of the centromere. Analysis of the offspring of doubly heterozygous females (i.e., for two of the loci—LDH-B, G6PDH, PEP-1, and PEP-2) shows that the four loci are independent.

The Jaylet test (newt micronucleus test) and the micronucleus test in xenopus: two in vivo tests on amphibia evaluation of the genotoxicity of five environmental pollutants and of five effluents

Abstract The sensitivity and the reliability of two in vivo genotoxicity tests were compared: the Jaylet test (or the newt micronucleus test) and the micronucleus test in xenopus. The Jaylet test is now a French standard method for testing genotoxicity in water, whereas the micronucleus test in xenopus was set up much more recently. Xenopus does, however, have certain practical advantages over the pleurodele. The study involved two phases, in the first we assessed the ability to detect the genotoxicity of five pure substances (selected for their environmental interest) added to the rearing water, and in the second we used the tests to evaluate genotoxicity of five effluents of urban and industrial origin. Three of the five tested substances (carbendazim, potassium chromate and cadmium chloride) were positive in the micronucleus test in xenopus but negative in the Jaylet test. One of these substances was positive with the Jaylet test but not with the micronucleus test in xenopus (pyrene). One substance (bis(tributyltin)oxide) gave negative results in both micronucleus tests. Only two of the five wastewaters were genotoxic with the Jaylet test: wastewater from a built-up area and effluent from a cokery. The effluent from a chemical plant was positive prior to the treatment stage. These two effluents give a positive response with the micronucleus test in xenopus. Wastewaters from the steel factory and the chemical plant were negative with the Jaylet test, but genotoxic to xenopus tadpoles.

Biomonitoring of the Genotoxic Potential of Draining Water from Dredged Sediments, Using the Comet and Micronucleus Tests on Amphibian (Xenopus Laevis) Larvae And Bacterial Assays (Mutatox® and Ames Tests)

Management of contaminated dredged sediments is a matter of great human concern. The present investigation evaluates the genotoxic potential of aqueous extracts of five sediments from French channels (draining water from dredged sediments), using larvae of the frog Xenopus laevis. Two genotoxic endpoints were analyzed in larvae: clastogenic and/or aneugenic effects (micronucleus induction after 12 d of exposure) and DNA-strand breaking potency (comet assay after 1 and 12 d of exposure) in the circulating blood. Additionally, in vitro bacterial assays (Microtox and Ames tests) were carried out and the results were compared with those obtained with larvae. Physicochemical analyses were also taken into account. Analytical analyses highlighted in the five draining waters a heavy load of contaminants such as metals and hydrocarbons. The results obtained with the micronucleus test established the genotoxicity of three draining waters. The comet assay showed that all 5 draining waters were genotoxic after 1 d of exposure. Although 3 of them were still genotoxic after 12 d of exposure, DNA damage globally decreased between d 1 and 12. The comet assay can be considered as a genotoxicity-screening tool. Data indicate that both tests should be used in conjunction in Xenopus. Bacterial tests (Ames) revealed genotoxicity for only one draining water. The results confirm the relevance of the amphibian model and the need to resort to bioassays in vivo such as the Xenopus micronucleus and comet assays for evaluation of the ecotoxicological impact, an essential complement to the physicochemical data.

Use of Aquatic Animals for Monitoring Genotoxicity in Unconcentrated Water Samples

The increasing quantity of pollutants in water is getting more and more concerning. Their origins are divers: industry, agriculture, domestic activities, but their ultimate destination is aquatic environment. The general toxicity of many of these pollutants, being usually spectacular and immediate, is easily detected. On the other hand, because the risk related to genotoxicity is less apparent and often delayed, it is consequently more insidious. The presence of genotoxins, even in low doses, does concern non aquatic species by the mean of the food chain and drinking water as well as aquatic ones. Therefore, their detection becomes an issue of prime importance for public health.

Evaluation of the genotoxicity of n-nitrosoatrazine, n-nitrosodiethanolamine and their precursors in vivo using the newt micronucleus test

Abstract The impact of amines and/or potential nitrosating agents (nitrite and nitrate) present in water was studied in the amphibian Pleurodeles waltl (urodele). The genotoxic effects of secondary amines, atrazine (AT) and diethanolamine (DEIA), alone or in combination with sodium nitrite or sodium nitrate, were evaluated on peripheral erythrocytes using the newt micronucleus test. At the maximum concentration (MC) which could be tested, neither AT (0.3 ppm) nor DEIA (75 ppm) were found to be clastogenic. Likewise, even at their respective MC, neither sodium nitrite (140 ppm) nor sodium nitrate (8000 ppm) induced the formation of micronuclei. Negative results were systematically obtained under all conditions examined (pH 8, 6 or 5; alternation or indirect natural daylight and darkness or continuous darkness; prior incubation of both precursors in the dark for 48 h). The genotoxic effects of N -nitrosamines suspected to be formed from such precursors were also evaluated. Larvae were reared in water containing non-toxic concentrations of N -nitrosoatrazine (NAT) (3.75, 7.5 and 15 ppm) or N -nitrosodiethanolamine (NDEIA) (6.25, 12.5, 25 and 50 ppm). We found a significant difference in level of micronucleated erythrocytes between the lowest (3.75 ppm) and highest (15 ppm) concentrations of NAT. With NDEIA, a dose-response relationship was observed at concentrations from 12.5 to 50 ppm. We suggest that at the concentrations used (close to those which may be encountered in a polluted natural aquatic environment), if NAT or NDEIA are formed, the amounts produced are probably too low to yield a positive response in the newt micronucleus test.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele, Amphibian). III. The relationship between sex-linked peptidase-1 expression and gene-dose effects

Erythrocyte peptidase-1 was analyzed by electrophoresis in various types of triploid Pleurodeles waltlii. Densitometric analysis of the zymograms showed (1) the existence of a gene-dose effect and (2) the presence of two Pep-1B alleles for one Pep-1A allele in heterozygous triploid females of biparental origin. Owing to the sex linkage of the PEP-1 locus (alleles Pep-1A and Pep-1B situated on the Z and W sex chromosomes, respectively), the results show that the sex genotype of these females is ZWW. In a particular line called series 103, the existence of a null allele was demonstrated. Densitometric analysis of females which were Pep-1A/Pep-10 (ZW), Pep-10/Pep-10 (WW), and Pep-1A/Pep-10/Pep-10 (ZWW) confirmed the gene-dose effect.

A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele Amphibian). I. Lactate dehydrogenase-B and glucose-6-phosphate dehydrogenase

On starch gel electrophoresis of erythrocyte hemolysates of Pleurodeles waltlii (Urodele Amphibian), both lactate dehydrogenase-B (LDH-B) and glucose-6-phosphate dehydrogenase (G6PDH) show polymorphism that depends on a pair of autosomal codominant alleles, confirmed by analysis of gynogenesis progeny. Diploid gynogenesis results from fusion of the female pronucleus with the second polar body. The heterozygous state of a female for a given character is maintained in certain progeny when crossing-over occurs between the locus in question and the centromere. So the high proportion of heterozygotes (45.7% for LDH-B and 76% for G6PDH) indicates the high frequency of crossing-over and hence the large distance between each of the loci and the centromere.

Maternal effect and embryonic gene expression for lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase (G6PDH), and peptidase (PEP-1) during the development of Pleurodeles waltl (urodele amphibian)

The polymorphism of three enzymes [lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase, (G6PDH), and peptidase-1 (PEP-1)] in Pleurodeles waltl has allowed the expression of the corresponding loci to be followed during the development of spawnings arising from various crosses. A maternal effect lasting up to the late tail-bud stage (approx. stage 28) was shown for PEP-1. A similar situation was observed for LDH-B and G6PDH. The embryonic alleles present retarded expression: from about stage 28 for PEP-1 and G6PDH and from about stages 22 to 24 (the young tail-bud stage) for LDH-B.

[Chromosome studies of Tylototriton verrucosus Anderson and of viable hybrids from Pleurodeles waltlii (female) x Tylototriton verrucosus (male) (urodele amphibians, Salamandridae)].

The karyotypes of T. verrucosus and of the viable intergeneric hybrid between P. waltlii ♀xT. verrucosus ♂ have been made up from different kinds of colchicinic metaphases (somatic larval cells and spermatogonia). T. verrucosus has no pair of telocentric chromosomes corresponding to the description previously made on another species, T. andersoni. — In T. verrucosus, a particular chromosome generally does not exhibit much morphological variability according to its origin, from a spermatogonial or a somatic cell. The chromosomes of T. verrucosus (2n=24) can be classified into three groups, each group being made of four pairs, like those of P. waltlii. The mean relative lengths and arm-ratios are nearly identical in both species for most of the chromosomes, except Nos. (2), (6) and (12): in T. verrucosus, the arm-ratio is higher than in P. waltlii for somatic chromosomes (2) and (6), and for gonial chromosomes (6), but is lower for somatic chromosomes (12). A few differences between the two species can be seen in the position of secondary constrictions which appear on somatic chromosomes after a cold treatment: in T. verrucosus, one constriction on each arm of chromosome (3), no constriction on the long arm of No. (6), one constriction on the short arm in most of the chromosomes (7), no constriction on No. (12). — In hybrid metaphases, chromosome (6) from Tylototriton is the only one which can be identified with certainty without a cold treatment. When secondary constrictions are present, it is possible to distinguish between the two components of pairs (3), (12) and occasionally (7), the two chromosomes (6) being always characteristic. — From triploid hybrid metaphases, it seems that the absolute length of individual chromosomes of T. verrucosus could be greater than that of homologous maternal chromosomes. — Problems of compatibility between the genetic characters of these related but geographically separated genera are shortly discussed.