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Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. References

Pro-oxidative effect of phenolic antioxidant (vitamin E) in combination with the initiators on human low-density lipoprotein is known. Recently I reported that oxidative stress induced by vitamin E in combination with the herbicide paraquat enhances structural chromosomal damage in cultured anuran leukocytes. In the present study, the phenolic antioxidant vitamin E-synthetic-analogue 2,6-di-tert-butyl-p-cresol (BHT) in combination with paraquat was found to enhance structural chromosomal damage in cultured Pelophylax (Rana) nigromaculatus leukocytes more than paraquat only and paraquat plus nicotinamido adenine dinucleotido phosphate served as positive control, although BHT only had no effect on induction of structural chromosomal damage. Paraquat plus BHT-enhanced structural chromosomal damage was inhibited by combination of the superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin and the hydrogen peroxide scavenger catalase. In test based on reduction of paraquat cation, BHT was found to reduce paraquat cation chemically to paraquat monocation radical. These results suggest that BHT functions in chemically donating electron to paraquat and thereby induces an acute accumulation of reactive oxygen species, resulting in increase in chromosomal damage.

Phenolic antioxidants including vitamin E and BHT are a group of natural and synthetic compounds to inhibit unsaturated fatty acid autoxidation-process promoted by free radical chain reaction (Březina et al. 1990; Frankel 1991; Wolf 2005). BHT is vitamin E synthetic analogue and shows little evidence regarding carcinogenic and mutagenic potential (Williams et al. 1999; Bomhard et al. 1992). In addition, BHT has the same antioxidant effect as vitamin E on lipidic free radical (Březina et al. 1990; Frankel 1991; Wolf 2005), and therefore widely used as antioxidant for cosmetics, foods, pet foods, plastics, hand-washing soap, rubber etc.

Previous reports have shown that vitamin E in combination with the initiators induces pro-oxidative effect on human low-density lipoprotein (Bowry et al. 1992; Kontush et al. 1996; Upston et al. 1999). Furthermore, vitamin E was shown to function as an electron donor to paraquat (PQ), which induces more reactive oxygen species (ROS) generation causing chromosomal damage (Hanada 2011). On the other hand, pro-oxidative action of BHT in combination with ‘redox-reaction-disruptor’ such as PQ has not been reported. The purpose of the present study is to investigate the possible mechanism of cytogenetic damage enhancement by BHT in combination with PQ.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. References

Chemicals

Paraquat (PQ) was purchased from Supelco (West Chester, USA). Penicillin–streptomycin solution, phytohemagglutinin M (PHAM), MEM amino acids solution 50× and MEM vitamin solution 100× were obtained from Invitrogen Co. Ltd., San Diego, California, USA. Other chemicals were obtained from Sigma-Aldrich, Inc. (St. Louis, Mo, USA). BHT was solubilized in ethanol.

Animal maintenance

Two mature male frogs (anuran species, Pelophylax (P.) nigromaculatus) were used in the present investigation. Specimens were derived from standard strain maintained in the Institute for Amphibian Biology, Graduate School of Science, Hiroshima University. Frogs were treated according to the basic principles expressed in Canadian Council on Animal care, guidelines on: the care and use of wildlife (2003). Specimens of P. nigromaculatus were fed on live crickets and raised at room temperature (26–27°C) (Kashiwagi et al. 2005). Maximum volume of blood collected from one frog (not sacrificed) is only approximately 500 μl. Therefore I designed this experiment, shown below. 1) Blood cells derived from one frog of the two frogs was applied in treatments of control, group 1, 3 and 4, and 2) those derived from the other frog was applied in treatments of group 2 and 5. Each group treatment is mentioned in ‘Genotoxic effect of PQ plus BHT on P. nigromaculatus leukocytes’ in ‘RESULTS’ section below for further detail.

Culture

Blood samples were collected from frogs anesthetized with diethyl ether and then incubated at 25°C in 1 ml of 70% Hank’s balanced salt solution (pH 7.2) containing 630 mg 1−1 lactose, 24.0 mg 1−1 L-glutamine, 0.2% (v/v) MEM amino acids solution 50×, 0.1% (v/v) MEM vitamin solution 100×, 11.8 mg 1−1 succinate, 10% fetal bovine serum (inactivated at 80°C for 5 min), 2 IU ml−1 heparin sodium, 1 μl ml−1 PHAM, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin for 5 days (Hanada 2002, 2011). Each volume of blood per 1 ml of the basal medium was approximately 20 μl.

Chromosome preparation

Chromosome preparations were made according to the method of Hanada (2002, 2011). Briefly, after treating with colchicine (final concentration 0.5 μg ml−1) for 4 h, culture mediums were removed. And then blood samples were transferred to each centrifuge tube containing 1 ml of 0.075 M potassium chloride solution and incubated for 20 min at 25°C. After fixation in 200 μl of 1:3 acetic acid/methanol fixative, blood samples were centrifuged at 266× g for 5 min. One ml of new fixative was poured into centrifuge tubes after supernatant removal. Obtained cell suspensions of blood samples were stored at –20°C until use. Chromosome preparations made using the air-drying method were incubated in 4% Giemsa solution in sodium phosphate buffer solution at pH 6.8 for 5 min and observed using light microscopy Axioscope 2 plus (ZEISS Co. Ltd.). Photographs of metaphase chromosomes were recorded using the digital camera Nikon D80. 100 metaphase figures were analyzed for each treatment. Figures of aberrant chromosomes in the figures in recent reports (Veličković 2004; Hanada 2011) were referred to in order to detect aberrant chro mosomes in cultured P. nigromaculatus leukocytes.

PQ cation (PQ2+) reduction test

One μl of 10−1 M BHT was added to 1 ml of 0.9 M Tris-HCl buffer solution (pH 7.2) containing 10−2 M PQ at 25°C, as previously described (Calderbank and Yuen 1965; Weidauer et al. 2002). Tests were conducted under anaerobic conditions induced by replacing air with nitrogen gas. PQ monocation radical (PQ+•) formation was spectrophotometrically monitored at 396 nm using UVIDEC-320H (Japan Spectroscopic Co. LTD). PQ+• concentration was calculated from absorbance at 396 nm using alkali sodium dithionite-formed PQ+• as the standard.

Statistical analysis

According to the method of Nicotera et al. (1985), 100 metaphase cells were analyzed per each group. Percentage of aberrant cells including damaged chromosome(s) was calculated as shown below.

Percentage (%) of aberrant cells = 100 × number of metaphase cells including damaged chromosome(s)/100 metaphase cells scored per each group.

Data were analyzed by χ2-test. p-values below 0.05 are considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. References

Normal karyotype and aberrant karyotype

Figure 1 shows the normal Pelophylax nigromaculatus karyotype and the aberrant karyotypes observed in group 4 leukocytes (10−6 M PQ + 10−4 M BHT treatment, see ‘Genotoxic effect of PQ plus BHT on P. nigromaculatus leukocytes’ in ‘RESULTS’ section below for further details). Figure 1A shows normal karyotype. Karyotype of P. nigromaculatus consists of five pairs of large chromosomes and eight pairs of small chromosomes. Second constriction is examined in center portion of long arms of no. 11 chromosome. Figure 1B and 1C show the aberrant karyotype in which the chromosomal break at proximal region of long arm of no. 4 and the chromosomal break (chromatid break) at distal portion of long arm of no. 1 is examined. In Fig. 1B, second constriction and fragile site have not been examined in proximal region of long arm of no. 4. Empty space region observed in the proximal region of long arm of no. 4 is not normal. In Fig. 1C, the length of left sister chromatid of aberrant no. 1 chromosome is slightly longer than that of right sister chromatid, because the left sister chromatid was elongated by chromatid break. The karyotype in Fig. 1C is also abnormal. Main chromosomal aberrations observed in control leukocytes and experiment group leukocytes were chromosomal break including chromatid break and isochromatid break as shown in Fig. 1B and 1C.

image

Figure 1. Normal karyotype of P. nigromaculatus (A) and aberrant karyotype of P. nigromaculatus (B and C). Arrows show break points in no. 1 and no. 4 chromosomes.

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Genotoxic effect of PQ plus BHT on leukocytes

Figure 2 shows the enhanced genotoxic effect of PQ plus BHT on leukocytes of P. nigromaculatus. Group-1 leukocytes were incubated in medium containing 10−6 M PQ for 6 h, and group-4 leukocytes were incubated in medium containing 10−6 M PQ and 10−6 to 10−4 M BHT for 6 h. Group-2 leukocytes (positive control) were incubated in medium containing 10−6 M PQ and 10−4 M NADPH for 6 h. Group-3 leukocytes were incubated in medium containing 10−6 to 10−4 M BHT for 6 h. Group-5 leukocytes were incubated in medium containing 10−6 M PQ, 10−4 M BHT, 1 μg ml−1 Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (Mn(III)TMpyP) and 0.1 U ml−1 catalase for 6 h. Untreated control leukocytes were incubated in medium and not exposed to BHT, Mn(III)TMpyP, catalase, NADPH or PQ. A concentration of 10−6 M PQ only induced the increase in the frequency of leukocytes including structurally aberrant chromosomes by 23%. At 10−6 M PQ plus 10−4 M NADPH significantly increased the frequency of the aberrant leukocytes by 44%. At 10−6 to 10−4 M BHT only had no effect on genotoxic damage, but 10−6 M PQ plus 10−4 M BHT induced the increase in the frequency of the aberrant leukocytes more than PQ only and PQ plus NADPH. The 10−6 M PQ plus 10−4 M BHT-enhanced genotoxic effect was significantly suppressed by dual inhibitory action of the radical scavengers, Mn(III)TMpyP and catalase. These results show that BHT functions in donating electron to PQ and then enhances generation of ROS, leading to chromosomal damage.

image

Figure 2. Effect of PQ plus BHT on P. nigromaculatus leukocytes. aSignificantly greater than corresponding value for untreated leukocytes. bSignificantly greater than corresponding value for group 1 (PQ only) leukocytes. cSignificantly less than corresponding value for group 4 (10−6 M PQ + 10−4 M BHT) leukocytes. dSignificantly greater than corresponding value for group 1 (PQ only) leukocytes.

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PQ+• formation by BHT

Vitamin E antioxidant-function-disruption has been previously shown to be induced by PQ (Hanada 2011). This vitamin E function-disruption seems to be involved in formation of free radical derived from vitamin E. It is, however, difficult to examine whether vitamin E reduces PQ2 +  chemically to PQ+• or not, because of insolubility of vitamin E in water. In the present study, therefore PQ+• formation-test was conducted using BHT that is more soluble in water than vitamin E, in order to confirm the hypothesis.

The PQ+• concentration induced by 10−4 M BHT was over 10−7 M, as shown in Fig. 3. The result shows that BHT chemically reduces PQ2 + to PQ+•.

image

Figure 3. PQ+• formation induced by BHT. Values given represent the mean value of three repetitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. References

Paraquat (PQ) is thought to enhance ROS generation after PQ+• formation induced by electron taken away from NADPH (Gage 1968; Dodge and Harris 1970; Bus et al. 1974), and then to induce chromosomal aberrations, as shown below (Nicotera et al. 1985; Tanaka and Amano 1989):

PQ2+  + NADPH (enzymatic reaction) [RIGHTWARDS ARROW] PQ+• formation [RIGHTWARDS ARROW] electron transfer to molecular oxygen [RIGHTWARDS ARROW] superoxide generation [RIGHTWARDS ARROW] superoxide dismutase reaction [RIGHTWARDS ARROW] hydrogen peroxide generation [RIGHTWARDS ARROW] hydroxyl radical generation by Fenton reaction [RIGHTWARDS ARROW] lipid peroxidation [RIGHTWARDS ARROW] induction of chromosomal aberrations

In the present study, PQ induced more structural chromosomal damage than untreated control, suggesting that PQ causes the chromosomal damage through ROS generation. Furthermore, PQ plus NADPH (positive control) induced more structural chromosomal damage than PQ only. These results suggest that PQ deprives electron of NADPH and then forms more PQ+• enhancing chromosomal damage.

BHT antioxidant-function is to inhibit formation of lipidic free radicals resulted from unsaturated fatty acid autoxidation (Březina et al. 1990; Frankel 1991). Theoretical mechanism model of BHT antioxidant-function is a belief that PQ-induced lipidic radical formation is suppressed by BHT and then decreases frequency of structural chromosomal damage. But, in the present study, BHT in combination with PQ increased the frequency of chromosomal damage more than PQ only and PQ plus NADPH, regardless of the result which BHT only has no effect on induction of structural chromosomal damage. The results show that BHT functions as electron donor to generate PQ+• . In addition, this suggestion is also supported by the result of PQ+• formation-test conducted in the present study. The chromosomal damage acutely increased by PQ plus BHT was inhibited by combination of the radical scavengers, Mn(III)TMpyP and catalase. Mn(III)TMpyP has superoxide dismutation ability to catalyze conversion of •O2 into H2O2 (Pasternack et al. 1981), and catalase is a crucial enzyme that catalyzes decomposition of H2O2 into water and molecular oxygen. This dual inhibition by the radical scavengers is more effective than the inhibition by each radical scavenger (Hanada 2011). These results suggest that BHT in combination with PQ enhances PQ+• formation, thereby increasing ROS generation and inducing more structural chromosomal damage.

Pro-oxidative action of BHT in combination with PQ on cultured P. nigromaculatus leukocytes is very similar to the vitamin E function-disruption-action by PQ. Chemical PQ2+  reduction by vitamin E and BHT, however, has not been reported until today. According to Hanada (2011), PQ-induced vitamin E function- disruption should occur because of unpaired electron of tocopheroxyl radical generated after hydrogen transfer reaction (one of the defense systems by phenolic antioxidants) of vitamin E. Disruption of BHT antioxidant- function may be caused by instability of unpaired electron of BHT free radical generated after hydrogen transfer reaction as well.

References

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. References