ECOTOXICOLOGY
AND
ENVIRONMENTAL
Monitoring
SAFETY
3, 122-125
(1979)
of Genetic Environmental Mutagenicity Tests1
Risk with New
H. W. RENNER Institute
of Biochemistry, 20, D-7500
Federal Karlsruhe, Received
Research Federal August
Centre for Nutrition, Republic of Germany
Engesserstr.
16, 1978
Genetic toxicology is a relatively new subdiscipline of toxicology. During recent years many mutagenicity tests have been developed to detect mutagenic substances, and these are based on different genetic effects. A single test can only offer information on one particular aspect of the possible mutagenic damage. Therefore, several different tests should be carried out in order to cover the variety of possible mutagenic effects which can occur at the genetic and chromosomal level. A mutagen is a substance which is able to induce mutations. It causes permanent heritable changes in the genetic material of the organism (DNA). Mutagenicity tests range from cytogenetic investigations on plant roots, yeast cells, and special bacterial strains to tests on Drosophila, cell cultures, and mammals. For screening purposes the Salmonella liver microsome test (the so-called Ames test) hasbecome accepted worldwide. In general mutagenicity tests are divided into in vitro and in vivo tests. Those mutagenicity tests which include metabolism, such as mammalian tests, are considered to be the most relevant. Two very interesting and important intermediate tests are the host-mediated assay (where test bacteria are incorporated into the peritoneum of small mammals for several hours) and the human lymphocyte cultures. In the latter case the problem of extrapolation of the results from animal tests to man does not arise. In the following I shall outline some of the most important mammalian tests. To be just, I should say that those tests which detect mutagenic effects on the germ cells are most important. But mutagenicity investigations on somatic cellsfor example, bone marrow cells-provide us with additional information about the carcinogenicity of a compound. Today we know that more than 90% of all carcinogens are also mutagens. This close correlation between carcinogenesis and mutagenesis suggests that damage to the genome is involved in both effects. This is a very important aspect not only in medicine but also in environmental toxicology. The most important mammalian tests are as follows: The dominant-lethal test gives us a measure of the cytogenetic effects induced via germ cells resulting in increased mortality of the embryos as a consequence of lethal mutations. 1 Paper presented at the Environmental Chemicals,”
meeting August
0147-6513/79/020122-04$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
on “Scientific Basis 16- 18, 1978, Vienna. 122
for
the
Ecotoxicological
Assessment
of
NEW
MUTAGENICITY
TESTS
123
The most suitable laboratory test animal is the mouse. In general, treated males are paired with untreated females. The matings start following mutagenic treatment and continue during the whole period of spermiogenesis (=7 weeks). Between the 14th and 16th day of pregnancy the females are sacrificed, the genitals with ovaries removed, and the number of dead implants of the test group compared with that of the controls. Four parameters are recorded: corpora lutea, preimplantation losses, dead implants, and live embryos. High numbers of animals are necessary in orderto obtain results which can be interpreted statistically. The micronucleus test is a cytogenetic screening test, carried out on bone marrow cells of small animals. Micronuclei are chromatid fragments of damaged cells which are not incorporated into the nuclei of daughter cells following mitotic cell division. In hematology they are well known as “Jolly bodies.” They are found in different cell types of the bone marrow but are best recognizable in the early-formed polychromatic and normochromatic erythrocytes. After the last cell division the erythroblasts expel their nuclei but the micronuclei remain in the cytoplasm of the erythrocytes. They are readily visible after Giemsa staining. The number of micronucleated polychromatic erythrocytes increases with higher doses of the mutagen. The chromosomal aberration tes? is a bone marrow test also. It detects numerical and structural aberrations and indicates the specific type of chromosomal damage. The Chinese hamster is the favored animal for this test because it has a low number of chromosomes (12 = 22) and the chromosomes are easily distinguishable from each other. The femur bone marrow cells of the colchicine-pretreated animals are rinsed, treated with a hypotonic solution, fixed with methanol/acetic acid (3:1), spread, and stained with 2% aceto-orcein solution. The slides are then covered and mounted. The sister chromatid exchange test (abbreviated “SCE”) has been used for some time in cell culture. Since 1976 this test system has also been carried out as an in vivo method and since the beginning of this year the methodology has been extensively standardized. Treatment of the living animals with bromodeoxyuridine, an analog of thymidine, leads, after two cell cycles (about 25 hr) and special staining of the cell material obtained, to chromatids stained in a differential way. The SCEs can then be counted. The number of SCEs increases in proportion to dose after administration of a mutagen. SCEs are probably double-stranded events. They indicate damage in the chromosomal organization provoked prior to other types of damage. This test seems to be the most sensitive in vivo test system to detect mutagenic substances. This completes the brief survey of the most important mutagenicity tests currently in use. It may have given you the impression that these tests are relatively simple and clear-cut but needless to say there are a number of problems and difficulties involved. I would just like to present in summary form a short list of some of these: (1) The sensitivity of the test system; (2) The selection of reliable methodologies for assessing the mutagenic activity;
124
H. W. RENNER
(3) The interpretation of data and the extrapolation to man; (4) The extent of synergistic effects between different chemicals; (5) The role of comutagens (for example, caffeine has been recognized as a comutagen); and (6) The metabolic activation/detoxification of the compounds. In order to validate the in vivo SCE test we carried out a study recently with some cytostatics commonly used in medicine. All of these anticancer therapeutics are mutagens. They have different chemical structures or modes of action (for example, alkylating agents, antibiotics, etc.). Table 1 shows in the first column the generic and trade name of the cytostatics and in the two subsequent columns the toxicological values and therapeutic doses. The next four columns show the lowest genetically effective doses of these drugs in the most used tests. The last column indicates the lowest effective dose for the in vivo SCE test using the Chinese hamster. It is quite clear from these results that this test is about 10 times more sensitive than all previously used tests. Indeed, in the case of Trenimon the sensitivity is increased 600-fold. In our opinion this represents a big step toward greater safety for the population in terms of medicines, consumption of foods contamiTABLE GENETIC
TOXICOLOGY
1
OF SOME Lowest
Substance
Toxicol. value bdk)
Therapeut. dose: man b-W&W
Dominantlethal test
Micronucleus test
CYTOSTATICS effective
dose (mg/kg
Chromes. aberration test
body wt) In viva SCE test with Chin. hamsters
Other tests
Methylmethanesulfonate (reference substance)
LD,: 2 x 106 (mice)
-
10 (mice)
20 (mice)
Cyclophosphamide (Endoxan)
LD,: 2 x 138 (mice)
4-6
40 (mice)
2 x 10 (mice)
9 (rats)
Metaphase li Oocytes > 50 (mice)
1
Triaziquone (Trenimon)
LD,: 2 x 0.14 (mice)
0.1-0.5
0.125 (mice)
0.062 (Chin. hamster)
2 x 0.031 (Chin. hamster)
Cultured lymphoc. (man) O.OOC25 mg/liter (cone) Chrom. aberr.
0.0001
LD,: 2 x 2.33 LD,,: 8 (mice)
0.025
5.25 (mice)
0.35 (mice) 0.5 (rhesus)
1 (rhesus)
Spermatogon. test (mice)
0.1
2 x 250 (Chin. hamster)
2 x 250 (Chin. hamster)
Cultured lymphoc. 4 x 0.25
2 x 6.25 (mice)
100 (mice)
Mammal. spot test (mice) 5x2
Mitomycin
Bleomycin
Procarbacine (Natulan)
C
LD,,: 168 (rats) LD,,: 460 (mice)
0.1.5-0.25
0.6-4
100 (mice)
Translocat. (mice) 40
test
I
I
2.5 (man) IO
NEW
MUTAGENICITY
125
TESTS
Saccharrn
I FIG.
1. Induction
of sister
:
chromatid
exchanges
“’t.5
5
: I0
by saccharin
al’(a
(Chinese
hamsters.
bone marrow
cells).
nated with undesired small quantities of noxious compounds, and exposure to environmental poisons. On the topic “foods” and “environment” I shall finally mention briefly two recent results with the new in viva SCE test, obtained in our laboratory. (1) Saccharin: Many investigations show saccharin to be a weak mutagen in bacterial systems, in Drosophila, and in cell cultures when tested using high doses. For mammalian systems positive, doubtful, and negative results have been reported. Figure 1 shows the result of the in vivo SCE test with saccharin. The dose of 5 g/kg of -body weight of saccharin used in our experiment should be seen in relation to the acceptable daily intake values of 5- 15 mg/kg for diabetics as determined by the FAO/WHO expert committee. This is a safety factor of about 1:500 or 1:1050, respectively. Certain impurities of saccharin such as o-toluenesulfonamide and their toxicological relevance should be taken into account. (2) 2,4,5-Trichlorphenoxyacetic acid (2,4,5-T) is a herbicide. In Vietnam it was widely employed for defoliation purposes. This compound has often been examined for mutagenicity. To our knowledge positive results have only been found in nonmammalian systems. Therefore this herbicide is classified with regard to its possible mutagenic activity as “uncertain.” Figure 2 shows the result of the in vivo SCE test with this compound. The curve reflects a slight mutagenic effect of the 2,4,5-T. On the basis of this example of a herbicide it can be demonstrated that the in viva SCE test is a highly sensitive tool to detect mutagens in our environment also. 1 2.WTrichlorphenoxy
acetic acid
control
FIG. 2. Induction of sister hamsters, bone marrow cells).
chromatid
exchange
by
2,4,5&chlorophenoxyacetic
acid
(Chinese
AND
ENVIRONMENTAL
Monitoring
SAFETY
3, 122-125
(1979)
of Genetic Environmental Mutagenicity Tests1
Risk with New
H. W. RENNER Institute
of Biochemistry, 20, D-7500
Federal Karlsruhe, Received
Research Federal August
Centre for Nutrition, Republic of Germany
Engesserstr.
16, 1978
Genetic toxicology is a relatively new subdiscipline of toxicology. During recent years many mutagenicity tests have been developed to detect mutagenic substances, and these are based on different genetic effects. A single test can only offer information on one particular aspect of the possible mutagenic damage. Therefore, several different tests should be carried out in order to cover the variety of possible mutagenic effects which can occur at the genetic and chromosomal level. A mutagen is a substance which is able to induce mutations. It causes permanent heritable changes in the genetic material of the organism (DNA). Mutagenicity tests range from cytogenetic investigations on plant roots, yeast cells, and special bacterial strains to tests on Drosophila, cell cultures, and mammals. For screening purposes the Salmonella liver microsome test (the so-called Ames test) hasbecome accepted worldwide. In general mutagenicity tests are divided into in vitro and in vivo tests. Those mutagenicity tests which include metabolism, such as mammalian tests, are considered to be the most relevant. Two very interesting and important intermediate tests are the host-mediated assay (where test bacteria are incorporated into the peritoneum of small mammals for several hours) and the human lymphocyte cultures. In the latter case the problem of extrapolation of the results from animal tests to man does not arise. In the following I shall outline some of the most important mammalian tests. To be just, I should say that those tests which detect mutagenic effects on the germ cells are most important. But mutagenicity investigations on somatic cellsfor example, bone marrow cells-provide us with additional information about the carcinogenicity of a compound. Today we know that more than 90% of all carcinogens are also mutagens. This close correlation between carcinogenesis and mutagenesis suggests that damage to the genome is involved in both effects. This is a very important aspect not only in medicine but also in environmental toxicology. The most important mammalian tests are as follows: The dominant-lethal test gives us a measure of the cytogenetic effects induced via germ cells resulting in increased mortality of the embryos as a consequence of lethal mutations. 1 Paper presented at the Environmental Chemicals,”
meeting August
0147-6513/79/020122-04$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
on “Scientific Basis 16- 18, 1978, Vienna. 122
for
the
Ecotoxicological
Assessment
of
NEW
MUTAGENICITY
TESTS
123
The most suitable laboratory test animal is the mouse. In general, treated males are paired with untreated females. The matings start following mutagenic treatment and continue during the whole period of spermiogenesis (=7 weeks). Between the 14th and 16th day of pregnancy the females are sacrificed, the genitals with ovaries removed, and the number of dead implants of the test group compared with that of the controls. Four parameters are recorded: corpora lutea, preimplantation losses, dead implants, and live embryos. High numbers of animals are necessary in orderto obtain results which can be interpreted statistically. The micronucleus test is a cytogenetic screening test, carried out on bone marrow cells of small animals. Micronuclei are chromatid fragments of damaged cells which are not incorporated into the nuclei of daughter cells following mitotic cell division. In hematology they are well known as “Jolly bodies.” They are found in different cell types of the bone marrow but are best recognizable in the early-formed polychromatic and normochromatic erythrocytes. After the last cell division the erythroblasts expel their nuclei but the micronuclei remain in the cytoplasm of the erythrocytes. They are readily visible after Giemsa staining. The number of micronucleated polychromatic erythrocytes increases with higher doses of the mutagen. The chromosomal aberration tes? is a bone marrow test also. It detects numerical and structural aberrations and indicates the specific type of chromosomal damage. The Chinese hamster is the favored animal for this test because it has a low number of chromosomes (12 = 22) and the chromosomes are easily distinguishable from each other. The femur bone marrow cells of the colchicine-pretreated animals are rinsed, treated with a hypotonic solution, fixed with methanol/acetic acid (3:1), spread, and stained with 2% aceto-orcein solution. The slides are then covered and mounted. The sister chromatid exchange test (abbreviated “SCE”) has been used for some time in cell culture. Since 1976 this test system has also been carried out as an in vivo method and since the beginning of this year the methodology has been extensively standardized. Treatment of the living animals with bromodeoxyuridine, an analog of thymidine, leads, after two cell cycles (about 25 hr) and special staining of the cell material obtained, to chromatids stained in a differential way. The SCEs can then be counted. The number of SCEs increases in proportion to dose after administration of a mutagen. SCEs are probably double-stranded events. They indicate damage in the chromosomal organization provoked prior to other types of damage. This test seems to be the most sensitive in vivo test system to detect mutagenic substances. This completes the brief survey of the most important mutagenicity tests currently in use. It may have given you the impression that these tests are relatively simple and clear-cut but needless to say there are a number of problems and difficulties involved. I would just like to present in summary form a short list of some of these: (1) The sensitivity of the test system; (2) The selection of reliable methodologies for assessing the mutagenic activity;
124
H. W. RENNER
(3) The interpretation of data and the extrapolation to man; (4) The extent of synergistic effects between different chemicals; (5) The role of comutagens (for example, caffeine has been recognized as a comutagen); and (6) The metabolic activation/detoxification of the compounds. In order to validate the in vivo SCE test we carried out a study recently with some cytostatics commonly used in medicine. All of these anticancer therapeutics are mutagens. They have different chemical structures or modes of action (for example, alkylating agents, antibiotics, etc.). Table 1 shows in the first column the generic and trade name of the cytostatics and in the two subsequent columns the toxicological values and therapeutic doses. The next four columns show the lowest genetically effective doses of these drugs in the most used tests. The last column indicates the lowest effective dose for the in vivo SCE test using the Chinese hamster. It is quite clear from these results that this test is about 10 times more sensitive than all previously used tests. Indeed, in the case of Trenimon the sensitivity is increased 600-fold. In our opinion this represents a big step toward greater safety for the population in terms of medicines, consumption of foods contamiTABLE GENETIC
TOXICOLOGY
1
OF SOME Lowest
Substance
Toxicol. value bdk)
Therapeut. dose: man b-W&W
Dominantlethal test
Micronucleus test
CYTOSTATICS effective
dose (mg/kg
Chromes. aberration test
body wt) In viva SCE test with Chin. hamsters
Other tests
Methylmethanesulfonate (reference substance)
LD,: 2 x 106 (mice)
-
10 (mice)
20 (mice)
Cyclophosphamide (Endoxan)
LD,: 2 x 138 (mice)
4-6
40 (mice)
2 x 10 (mice)
9 (rats)
Metaphase li Oocytes > 50 (mice)
1
Triaziquone (Trenimon)
LD,: 2 x 0.14 (mice)
0.1-0.5
0.125 (mice)
0.062 (Chin. hamster)
2 x 0.031 (Chin. hamster)
Cultured lymphoc. (man) O.OOC25 mg/liter (cone) Chrom. aberr.
0.0001
LD,: 2 x 2.33 LD,,: 8 (mice)
0.025
5.25 (mice)
0.35 (mice) 0.5 (rhesus)
1 (rhesus)
Spermatogon. test (mice)
0.1
2 x 250 (Chin. hamster)
2 x 250 (Chin. hamster)
Cultured lymphoc. 4 x 0.25
2 x 6.25 (mice)
100 (mice)
Mammal. spot test (mice) 5x2
Mitomycin
Bleomycin
Procarbacine (Natulan)
C
LD,,: 168 (rats) LD,,: 460 (mice)
0.1.5-0.25
0.6-4
100 (mice)
Translocat. (mice) 40
test
I
I
2.5 (man) IO
NEW
MUTAGENICITY
125
TESTS
Saccharrn
I FIG.
1. Induction
of sister
:
chromatid
exchanges
“’t.5
5
: I0
by saccharin
al’(a
(Chinese
hamsters.
bone marrow
cells).
nated with undesired small quantities of noxious compounds, and exposure to environmental poisons. On the topic “foods” and “environment” I shall finally mention briefly two recent results with the new in viva SCE test, obtained in our laboratory. (1) Saccharin: Many investigations show saccharin to be a weak mutagen in bacterial systems, in Drosophila, and in cell cultures when tested using high doses. For mammalian systems positive, doubtful, and negative results have been reported. Figure 1 shows the result of the in vivo SCE test with saccharin. The dose of 5 g/kg of -body weight of saccharin used in our experiment should be seen in relation to the acceptable daily intake values of 5- 15 mg/kg for diabetics as determined by the FAO/WHO expert committee. This is a safety factor of about 1:500 or 1:1050, respectively. Certain impurities of saccharin such as o-toluenesulfonamide and their toxicological relevance should be taken into account. (2) 2,4,5-Trichlorphenoxyacetic acid (2,4,5-T) is a herbicide. In Vietnam it was widely employed for defoliation purposes. This compound has often been examined for mutagenicity. To our knowledge positive results have only been found in nonmammalian systems. Therefore this herbicide is classified with regard to its possible mutagenic activity as “uncertain.” Figure 2 shows the result of the in vivo SCE test with this compound. The curve reflects a slight mutagenic effect of the 2,4,5-T. On the basis of this example of a herbicide it can be demonstrated that the in viva SCE test is a highly sensitive tool to detect mutagens in our environment also. 1 2.WTrichlorphenoxy
acetic acid
control
FIG. 2. Induction of sister hamsters, bone marrow cells).
chromatid
exchange
by
2,4,5&chlorophenoxyacetic
acid
(Chinese
