Antimutagenic potentials of L-tyrosine and its metabolites on the genotoxic activity induced by methyl methanesulfonate

© 2017 The Authors. Published by IASE. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).


Introduction
*In genetic toxicology, studying of the extent of DNA damage at the chromosome level is very important because it is an important phase in carcinogenesis. Micronucleus assay is an important tool in assessing the chromosome damage inasmuch as it reliably measure both the chromosome loss and chromosome damage (Fenech, 2000).
Alkylating agents can cause cell death, mutation and cancer, are substances that alter the structure of DNA and classified as genotoxic to both somatic cells and germ cells. The biological properties and mechanism of action of alkylating agents have been extensively explored in cells and animal model systems are categorized as mutagenic, toxic, clastogenic and teratogenic (Margison et al., 2002;Magee et al. 1976). Therefore, genotoxic and mutagenic substances in the environment must be evaluated and monitored as they often induce genetic toxicities and abnormalities.
Antimutagens are substances which can trap genotoxins and mutagens through molecular interaction which eventually inhibit the metabolism of promutagens (Sylianco, 1998). Amino acids are essential for the protein synthesis which is required for enzymatic catalysis, control growth and differentiation, transport, storage, protection, hormones, and regulation. Studies done by Bajo (1994) shows that L-Tyrosine and its metabolic product dopamine did not possess direct DNA damaging capacity on H17Rec + and M45Rec -strains of Bacillus subtilis. However, they were able to reduce the genotoxicity of quinoline. It has been shown from the studies of Sylianco (Sylianco and Guevara, 1985;Sylianco and Guevara, 1989;Sylianco, 1991) that amino acids, such as glutamic acid, aspartic acid, lysine, histidine, arginine, and cysteine; mineral ions such as calcium, magnesium, zinc, copper, iron, and manganese inhibit and reduce the activity of some mutagens and carcinogens such as dimethylhydrazine, benzo (a) pyrene, dimethylnitrosamine, azaserine and methyl methanesulfonate. In this study, the mutagenic and antimutagenic potential of L-tyrosine and its metabolites were evaluated using peripheral blood and bone micronucleus assays, and Ames test.

Dosage
In the micronucleus test, the experimental dosage of the genotoxic substance performed in the antimutagenecity study of Bajo (1994) were used for L-Tyrosine (30) and dopamine (300); for epinephrine (0.1), and L-DOPA (150) mg per kilogram body weight. For Ames test, L-Tyrosine (106.60 ppm), L-DOPA (509.43 ppm), dopamine (1,132.08 ppm), epinephrine (18.87 ppm) and MMS (37.74 ppm). There were two sets of control: Methyl methanesulfonate (10 mg/kg) as the positive control and distilled water as the negative control.

Test animals/microorganisms
Albino mice were purchased from a local petshop in Iligan City. They were fed with commercial pellets and distilled water throughout the acclimatization and experimental periods and were subjected to a regular light/dark cycle. The drinking bottles and cages were cleaned regularly. The animals were placed in a room at ambient temperature and caged in small groups according to feeding conditions. The test microorganisms Salmonella tester strains TA98 and TA104 were obtained from the Philippine National Collection of Microorganisms (PNCM), University of the Philippines-Los Baños, Laguna, Philippines with a permission from Dr. Bruce N. Ames.

The micronucleus test
Mice, both female and male, aged 8-12 weeks were randomly chosen. Each experimental group contained 5 mice. Prior to drug administration, mice were fasted from food and water for 16 hours and two hours after administration, food and water ad libitum were given to the experimental mice. The micronucleus assay was performed using peripheral blood and bone marrow simultaneously from the same animal with a double dosing regimen. L-Tyrosine, L-DOPA, dopamine and epinephrine; L-Tyrosine, L-DOPA, dopamine and epinephrine plus methyl methanesulfonate respectively were administered orally twice, each with 0.20 mL/20 gram body weight using a 500 uL microsyringe as an improvised gavage, 24 h apart, to five mice per dosage group. Both peripheral blood and bone marrow were collected 24 h after the second treatment.

Peripheral blood micronucleus assay
The method outlined in Aida et al. (1995), CSGMT (1990), Hayashi et al. (1983) and Heddle (1973) was followed with minor modifications. Prior to the collection of the blood, the mice were collapsed using chloroform and killed through cervical dislocation. Blood was collected by cutting the tail of the mouse and a drop of blood was placed on the pre-cleaned and coded glass slides, and smeared using a cover slip then air dried. The prepared blood films were fixed in methanol then air dried again. The smeared preparations were stained with acridine orange (AO).

Bone marrow micronucleus assay
The method outlined in Schmid (1976) was followed with modifications. The mice were sacrificed by cervical dislocation. The femora were excised and cleaned of muscle tissues present then the bone marrow was flushed with 0.20 mL fetal calf serum in a micro test tube. The suspensions were centrifuged at 1000 rpm for 5 minutes to sediment the cells; supernatants was discarded and each of the sediments were homogeneously mixed using pasteur pipette then smeared on the pre-cleaned and coded glass slides, and smeared using a cover slip. The prepared bone marrow films were air-dried and fixed in methanol then air dried overnight and was stained the following morning. Staining of slides were done in a undiluted May-Gruenwald stain for 5 minutes, then were transferred to a 50% May-Gruenwald stain for 2 minutes, after which, the slides were immediately transferred to a 15% aqueous Giemsa stain solution for 10 minutes then the back was wiped with a tissue paper.
Screening of the slides for micronucleated polychromatic erythrocytes in the peripheral blood and bone marrow cells followed using 1000x magnification. Five slides per mouse were prepared and scoring was done from 1000 cells per slide or 5000 per mouse.

Ames test
The method outlined in the studies conducted by Mortelmans and Zeiger (2000), Ames et al. (1973), and Maron and Ames (1983) was followed minor modification. To the sterile 20 mL test tube the following were added as follows: 0.5 mL of phosphate buffer, 0.1mL of bacterial culture, and 0.05 mL or less of test solution. The mixture was mildly mixed using vortex. The mixture was incubated for 20 minutes at 37 o C. After the incubation, 2 mL of top agar (with 0.5mM histidine/biotin solution) maintained at 43-48 o C was added. The contents of test tubes are then mixed using vortex at moderate speed for 3 seconds and poured onto the surface of minimal-glucose agar plates then tilted and rotated for even distribution of the mixture. The top agar was set aside to harden for an hour. When the top agar has hardened, the plates are inverted and placed in a 37 o C incubator. After 48-72 hours, the plates were removed from the incubator and number of visible colonies per plate was counted. Three trials per test sample were done with three plates per trial. Controls were done side by side with the test samples with three plates per trial. Methyl methanesulfonate and sterilized distilled water was used as the positive control and negative control, respectively.

Statistical evaluation
Statistical analyses of the results were undertaken using the One-way Analysis of Variance (ANOVA) with square root transformation of raw data and Duncan's Multiple Range Test (DMRT).

In vivo effects
Micronucleus assays using peripheral blood reticulocytes and bone marrow cells were used to evaluate the antimutagenic effects of the test compounds on the known alkylating and carcinogen, methyl methanesulfonate.
Results on Table 1 show that L-Tyrosine, L-DOPA, dopamine, and epinephrine lack the capacity of forming micronucleated polychromatic erythrocytes (MPCEs) which suggests a lack of chromosome breaking effects on the peripheral blood reticulocytes and bone marrow cells. These results indicate that the test samples do not alter the structure of DNA. The number of micronucleated erythrocytes found on these substances is mainly due to the spontaneous fragmentation of the chromatin material at the level of the negative control.
The positive control, methyl methanesulfonate (MMS), showed a very significant number of micronucleated polychromatic erythrocytes in comparison with the negative control, and L-Tyrosine and its metabolites.  Table 2 show that L-Tyrosine has the highest activity and epinephrine has the lowest activity in reducing the number of number of MPCEs in the peripheral blood micronucleus assay and in the bone marrow micronucleus assay, epinephrine and L-DOPA has the lowest activity in reducing the number of MPCE's and the activity of L-Tyrosine, L-DOPA, and dopamine reducing the number of MPCE's were insignificantly different. Generally, these results mean that L-Tyrosine and its metabolites can inhibit the chromosome breaking potential of MMS. The possible mechanism of the molecular interaction of the alkylating agent (CH3 + ) with DNA is shown Fig. 1. When guanine is alkylated at N-7 (Fig.  2), apurinic sites were produced and this will happen if repair is not carried out immediately.
These apurinic sites may lead to the destabilization of base pairs of the DNA. It has also been found that when alkylation takes place at O-6 ( Fig. 3) of the purine base, guanine, mispairs could induce destabilization of the DNA, leading to a chromosome breakage. Alkylation does not only weaken the hydrogen bonds between the guanine and cytosine base pairs but also weakens the N-C bonds glycosidic bond.  Another possible mechanism of action on trapping the carbocation of the alkylating agent by the test substances is through electrophilic substitution on the aromatic ring. The electron cloud of the pi system of the benzene ring of the test substances can attract the carbocation; hence substitution of the hydrogen atom can take place, thus preventing the carbocation (CH3 + ) from alkylating the N-7 and O-6 of the guanine base of the DNA (Fig. 8). The next following Figs. 9, 10, 11, and 12 will show the electrophilic substitution on the benzene ring of the respective test compounds. Generally the results show that L-Tyrosine has the highest inhibitory effects on the chromosome damaging capacity of methyl methanesulfonate both on the peripheral blood reticulocytes and bone marrow cells. It is also found that epinephrine has the lowest inhibitory effects among the test substances both in peripheral blood reticulocytes and bone marrow cells.
The low inhibitory activity of epinephrine may be accounted to the lack of carboxylate group on its structure and less availability of the lone pair in the amino group of the β-carbon.

In vitro effect
The effects of L-Tyrosine and its metabolites on the mutagenic activity induced by methyl methanesulfonate was assessed on the number of growth of revertants using Ames test utilizing TA98 and TA104 as its test microorganisms.
Overall, Table 3 shows that the test substances are significantly different from the positive control in both TA98 and TA104 strains which implies that the test compounds did not induced reversion of the histo his + strains. This suggests that the test compounds are not mutagenic. The test compounds did reduce and inhibit the number of revertants induced by methyl methanesulfonate both in the standard tester strains TA98 and TA104 as shown in Table 4. This suggests that the test compounds have antimutagenic potentials. As such, epinephrine accounted the lowest activity in reducing and inhibiting the number of revertants induced by MMS both in the standard tester strains TA98 and TA104.
Generally, L-tyrosine and L-DOPA in standard tester strain TA98, and L-tyrosine, L-DOPA, and dopamine in standard tester strain TA104 has the highest activity in reducing the number of revertants induced by MMS.

Conclusion
Indeed, the use of the amino acid L-Tyrosine and its metabolites, L-DOPA, dopamine and epinephrine, were proven to be an attractive, effective and promising approach in reducing the number of micronuclei and number of revertants induced by MMS. In general, L-Tyrosine and its metabolites have an antimutagenic potential.