Evaluation of the clastogenicity of water treated with sodium hypochlorite or monochloramine using a micronucleus test in newt larvae (Pleurodeles waltl).

We have previously described a micronucleus test using erythrocytes from larvae of the urodele amphibian Pleurodeles waltl (pleurodele). The test is based on a comparison of the levels of micronucleated erythrocytes in blood smears from larvae reared in water containing a clastogen, with the levels from larvae reared in purified water. We have employed this test to evaluate mutagenic activity of chlorinated or monochloraminated water devoid of all organic matter. (i) The level of micronuclei in erythrocytes was compared between a group of larvae reared for 12 days in chlorinated reconstituted ultrapure water treated with sodium hypochlorite, and a control group reared in just the reconstituted water. Sodium hypochlorite was added when both the food and medium were changed each day. Chlorine levels of 0.125 and 0.25 p.p.m. led to significant elevations of micronuclei. (ii) The possibility of indirect effects of chlorine through chemical interactions with the food were also investigated, using the following scheme: larvae were left for 3 h in chlorinated reconstituted ultrapure water and then placed in non-chlorinated water. Food was only introduced when they were transferred to the non-chlorinated water. This procedure was repeated for 12 consecutive days. Control larvae were reared in non-chlorinated water throughout this period. In this case results were also positive when the larvae were exposed for only 3 h to the chlorine (0.2 p.p.m. for 12 days) in the absence of food. (iii) This was the same as experiment 1 except that the water was chlorinated with monochloramine instead of sodium hypochlorite.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro metabolism of benzo(a)pyrene by the larva of the newt, Pleurodeles waltl.

1. The larva of the amphibian species, Pleurodeles waltl was shown to metabolize benzo(a)pyrene in vivo into a variety of oxidized products. 2. In vitro, BaP hydroxylase (AHH) activity was found in hepatic microsomes and postmitochondrial fractions from both larvae and adults of the pleurodele. 3. The clastogenic effect of BaP formation of micronuclei in the erythrocytes was shown to be related to the presence of BaP quinones in the tissues of the newt.

The micronucleus test in Xenopus: a new and simple 'in vivo' technique for detection of mutagens in fresh water.

The aim of this study was to extend the micronucleus test to Xenopus laevis to screen water for potential mutagens. Up to now, the most commonly used methods for screening fresh water are the bacterial tests (Ames tests). Ames tests are well validated, but are merely in vitro bacterial assays which can only detect point mutations whereas the micronucleus test is an in vivo assay using a vertebrate species, and thus is able to detect both chromosome and genome mutations. This study was designed to optimize the micronucleus test for Xenopus. Three different variables were investigated: (i) the effect of temperature; (ii) the effect of the stage of larval development; and (iii) the effect of varying or continuous exposure concentrations. In addition, a dose-response curve was established for eight concentrations of benzo[alpha]pyrene. The results indicate that the micronucleus test with Xenopus can be used to monitor water pollution.

Use of newt larvae for in vivo genotoxicity testing of water: results on 19 compounds evaluated by the micronucleus test.

The micronucleus test using erythrocytes of Pleurodeles waltl larvae (Amphibian, Salamandridae) was used to detect the genotoxic activity of various substances. Most of them were known (or suspected) to be mutagenic or carcinogenic in mammals. Larvae were reared in water containing either a test chemical (experimental group) or in control water and the levels of micronucleated red blood cells (RBCs) compared between the two groups. Aroclor 1254, butylated hydroxyanisole, phenobarbital and 12-O-tetradecanoyl-phorbol-13-acetate produced negative results, while acridine orange, benzo[alpha]pyrene, epsilon-caprolactam, cyclophosphamide, diethyl sulphate, epichlorhydrin, ethidium bromide, ethyl methanesulphonate, ethylene dibromide (dibromoethane), N-ethyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea, hexamethylphosphoramide, 3-methyl cholanthrene, pyrene and o-toluidine gave positive responses. The results were compared with published data from other tests used to detect the clastogenic or mutagenic properties of chemicals. We suggest that the newt micronucleus test could be used to monitor aquatic pollution, and/or for the quality control of drinking water. Pleurodele larvae may also prove to be of value for the detection of carcinogenic/clastogenic substances dissolved in water.

Genotoxicity and bioaccumulation of methyl mercury and mercuric chloride in vivo in the newt Pleurodeles waltl.

Larvae and embryos of the urodele amphibian Pleurodeles waltl were raised for several days in water containing low concentrations of methyl mercuric chloride (CH3HgCl) or mercuric chloride (HgCl2). Examination of squash preparations from treated embryos showed that both compounds induced chromosome breaks and c-mitosis. Examination of blood smears from the larvae demonstrated a higher level of micronucleated erythrocytes in the animals raised in the mercury-containing water than in controls raised in uncontaminated water. The bioaccumulation of both compounds was evaluated by determination of mercury levels in the larvae. After 12 days of treatment, concentration factors (concentration in the organism/concentration in the water) of approximately 1200 and approximately 600 were found for methyl mercury and mercuric chloride, respectively.

An antioxidant protects against the clastogenic effects of benzo[a]-pyrene in the newt in vivo.

The clastogenic effect of benzo[a]pyrene (B[a]P) was observed in newt larvae by the formation of micronuclei in circulating red blood cells (RBCs). The level of micronucleated RBCs depended on the concentration of B[a]P (0.025, 0.05 and 0.1 p.p.m.) in the water in which the animals were reared. The synthetic antioxidant, 2(3)-tert-butyl(4) hydroxyanisole (BHA), currently used as a food additive (E 320), was added to the water at concentrations of 0.5, 1.0 and 1.5 p.p.m. It reduced the clastogenic effect of B[a]P in the test larvae, with the most marked effect at a concentration of 0.5 p.p.m. The genotoxicity of B[a]P and the protective action of BHA in the newt are discussed.

Detection of mutagenicity in drinking water using a micronucleus test in newt larvae (Pleurodeles waltl).

We have previously described a micronucleus test using erythrocytes from larvae of the urodele amphibians Pleurodeles waltl (pleurodele) and Ambystoma mexicanum (axolotl). The test is based on a comparison of the levels of micronucleated erythrocytes in blood smears from larvae reared in water containing a clastogen, with the levels from larvae reared in purified water. Using larvae from the pleurodele, we have employed this test to evaluate mutagenic activity in drinking water. Groups of larvae were reared in tap water, while control groups were reared in tap water which had been filtered over sand and active carbon to remove micropollutants. Seven separate tests carried out between October 1985 and May 1986 all gave positive results of varying degree depending on the time of year. This test is therefore able to detect clastogens in normal drinking water. It could be used for quality control of drinking water during the various stages in the treatment of raw water without any requirement for prior extraction or concentration of micropollutants.

A new micronucleus test using peripheral blood erythrocytes of the newt Pleurodeles waltl to detect mutagens in fresh-water pollution.

A model micronucleus test system using peripheral blood erythrocytes from larvae of Pleurodeles waltl is described. The most suitable larval stage for testing chemical treatments was determined. Larvae were reared in water containing one of the 4 compounds: benzo[a]pyrene (BaP), ethyl methanesulphonate (EMS), diethyl sulphate (DES) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Response curves as a function of treatment duration over a period of 16 days were plotted for 3 different concentrations of the 4 compounds in order to optimize conditions for a low dose micronucleus test. This model can be used as a monitoring system for the detection of fresh water pollution and can also be employed for clastogen screening of chemical compounds. The test is sensitive, reliable and easy to use.

Induction of micronuclei in peripheral erythrocytes of axolotl larvae following in vivo exposure to mutagenic agents.

Previous work from this laboratory demonstrated the presence of micronuclei in erythrocytes from larvae of the urodele amphibian Pleurodeles waltl reared in water containing clastogenic substances. In order to investigate the generality of this finding, larvae from another urodele Ambystoma mexicanum (axolotl) were reared in water containing one of the two following compounds: benzo[a]pyrene (BaP) or ethylmethane sulphonate (EMS). The level of micronucleated erythrocytes on blood smears was compared with control samples from larvae reared in fresh water. The optimum larval stage for this test system was determined. The effects of the indirect mutagen (BaP), and the direct mutagen (EMS) were found to depend on both dose and exposure to the clastogen. Positive results were obtained for BaP after 8 days of treatment at a concentration of 0.025 p.p.m. After 10 days of treatment at a concentration of 0.1 p.p.m. numerous micronuclei were seen (greater than 250%). Positive results were also obtained with EMS after 8 days of treatment at a concentration of 24 p.p.m. At 62 p.p.m., positive results were found after 6 days of treatment, while at 124 p.p.m. positive results were found after only 4 days. The results with both these agents show that the axolotl holds promise as an in vivo test system for the detection of low concentrations of clastogens in an aquatic environment.

Micronuclei in red blood cells of the newt Pleurodeles waltl after treatment with benzo(a)pyrene: dependence on dose, length of exposure, posttreatment time, and uptake of the drug.

Aquatic larvae of the newt Pleurodeles waltl were exposed to different concentrations of benzo(a)pyrene (BaP) for various lengths of time. Frequencies of micronuclei in circulating erythrocytes were determined at different times after termination of the treatment. The incidence of micronuclei in larvae kept for 8 days in BaP-containing water displayed a marked increase with dose up to 0.075 ppm and a more gradual one with higher doses, reaching 158 per 1,000 at 0.75 ppm. The lowest dose at which a significant increase could be discerned was 0.01 ppm. Short periods of exposure, less than 2 days, did not result in a marked increase in micronuclei. Uptake and release was studied with tritiated BaP. Larvae concentrated BaP rapidly, attaining maximal levels after 12 hr. The ratio of radioactivity in larvae to that in an equivalent volume of surrounding water was about 200 independent of the amount of BaP added. Calf serum or bovine serum albumin added to the water lowered this ratio by competing for binding to BaP. Radioactive larvae placed in regularly renewed noncontaminated water lost 99% of the label after 100 hr. It is concluded that pleurodele larvae are a promising model for the detection of genotoxic activity in the aquatic environment.

The role of endoderm in blood cell ontogeny in the newt Pleurodeles waltl.

In order to determine the origin of blood cells we performed embryonic grafts of different portions of mesoderm from various locations between diploid and tetraploid embryos at the tail-bud stage. The tetraploid animals were the hosts. The size differences between tetraploid and diploid cells made identification possible by direct microscopic examination of blood smears. In a previous report we showed the important role of truncal anterior mesoderm in the genesis of blood cells. We now establish that this effect is brought about by the inductive capacity of the hepatic endoderm or by the fact that the environmental conditions are more appropriate for blood stem cell development, whereas in the absence of the hepatic endoderm the blood stem cells fail to appear. Grafting of hepatic anlage containing endoderm and mesoderm gives rise to numerous graft-derived blood cells which last throughout the life span of the hosts. The same result is obtained by grafting truncal posterior ventral or lateral mesoderm onto hepatic endoderm. Heterotopic grafting of truncal anterior mesoderm isolated from its underlying hepatic endoderm leads to the formation of only a few blood cells which last only during larval life. This demonstrates that the whole lateral and ventral truncal mesoderm is able to differentiate into blood cells when associated with hepatic endoderm.

Micronuclei in red blood cells of the newt Pleurodeles waltl Michah: induction with X-rays and chemicals.

Pleurodeles waltl, a typical long-tailed amphibian (Urodela) is proposed as a new animal for genetic toxicological studies. X-Rays and various clastogenic substances cause the formation of clearly visible micronuclei in the red blood cells (RBC). The proportion of cells with micronuclei was determined from blood smears of larvae after irradiation or after having been kept in water containing the substances to be studied. A dose-effect curve was established for X-irradiation. The 6 following substances were tested by this method: benzo[alpha]pyrene, carbaryl, N-nitrosocarbaryl, aziridine, caffeine and formaldehyde. Formaldehyde was the only substance tested that did not bring about formation of micronuclei in the RBCs. The results were compared with data already obtained by other methods of toxicology. This method should allow a cytogenetic test to be developed for the detection of clastogenic substances in aqueous media.

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.

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.

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.

Induction of triploidy in the newt Pleurodeles waltlii by heat shock or hydrostatic pressure: interpretation of the different types of ploidy using a chromosomal marker.

Eggs of diploid females of Pleurodeles waltii, fertilized artificially by waltlii sperm, have been submitted to increased temperature of hydrostatic pressure during the first hour of development. Of the resulting viable individuals about 95% were triploid, the remainder diploid or tetraploid. Other types of ploidy have been observed in abnormal embryos (n, 2n/4n, 5n, n/5n, 6n). Some of the treated eggs came from females with a marker chromosome (pericentric inversion). The karyotypes of the animals developing from such eggs confirm that triploidy results from retention of the second polar body and make it possible to interpret the origin of the other types of ploidy. From the results it can be envisaged how gynogenetic animals can be obtained from eggs inseminated by inactivated sperm and shock-treated to restore diploidy.

Experimental gynogenesis in the newt species Pleurodeles waltlii and P. poireti.

Eggs of diploid females of Pleurodeles waltlii, inseminated by genetically inactivated sperm of Salamandra salamandra, have been treated by heat-shock or increased hydrostatic pressure during the first hour of their development. The resulting viable gynogenetic individuals show different degrees of ploidy although they mostly are diploid. The use of females with a pericentric inversion as a marker chromosome allow the chromosomal constitution to be clarified. Eggs of females heterozygous for a recessive semilethal mutation (ascite caudale, ac) subjected to the same experimental treatment gave 50% ac/ac embryos. Experiments with Pleurodeles poireti gave the same results as those with P. waltlii. These observations prove that gynogenetic Urodeles can be produced in large numbers. In their offspring the detection of inherent or spontaneous recessive mutations is greatly facilitated.

Thymic lymphocyte origin in the newt Pleurodeles waltlii studied by embryonic grafts between diploid and tetraploid embryos.

Thymic lymphocytes origin in the newt Pleurodeles waltlii was studied by embryonic grafts of the gill area between diploid and tetraploid embryos. By this way, either cell size or chromosome number are used as markers. Heterotopic transplantation of the gill area to the ventral region of a host embryo is followed by differentiation of gills and gill-associated structures including thymus. Analysis of cell size and chromosome number shows that in Pleurodeles, thymocytes do not differentiate in situ but originate from blood-borne stem cells that migrate into the thymus anlage.

[Studies on the origin of various blood cell lines in the salamander Pleurodeles waltlii (author's transl)]. / Recherches sur l'origine des différentes lignées de cellules sanguines chez l'amphibien urodèle Pleurodeles waltlii

Grafting of haemopoietic organs was performed at several developmental stages between diploid and autotetraploid individuals of Pleurodeles waltlii. The difference in size between diploid and tetraploid cells is so obvious that their identification is possible by direct microscopic examination. To avoid immunological rejection, the grafts have been made with inbred animals. 2. After grafting the spleen, the host cells are observed to colonize it. This phenomenon is more important with spleens from donors at early stages of development. When the spleen from a late larval stage or a metamorphosed animal is transplanted into another larva, the blood of the latter contains 66 or 72% erythrocytes originating from the graft. 3. Embryonic liver grafting is followed by a colonization of all haemopoietic organs by cells originating from the granulopoietic tissue of the graft. The proportion of grafted blood cells is then very high. A liver graft performed at a larval stage gives qualitatively similar results but the numbers of cells originating from the graft are lower. A survey of intra-cardiac erythropoiesis in these animals shows that it develops from stream blood cells and not from cardiac endothelial cells. 4. After thymus graft, the lymphoid part of the organ is replaced by the host cells. 5. From these results, it is suggested that the granulopoietic liver tissue contains one or several kinds of stem cells which could differentiate into all types of blood cells.