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Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow
Toxicology Letters 93 (1997) 205 – 213
Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow G.C. Jagetia *, R. Aruna Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India Received 24 June 1997; received in revised form 6 October 1997; accepted 6 October 1997
Abstract The frequency of micronuclei (micronucleated polychromatic erythrocytes, MPCE and micronucleated normochromatic erythrocytes, MNCE) was studied at 12, 24 and 36 h post-treatment in the bone marrow of mice treated with 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt of hydroquinone (HQ). Treatment of mice with various doses of HQ resulted in a dose dependent increase in the frequency of both MPCE and MNCE at all the post-treatment time periods. The frequency of MPCE was significantly higher after administration of 3.125 mg/kg HQ at 24 h post-treatment, except 12 and 36 h, where a significant increase in the frequency of MPCE was observed only after administration of 6.25 mg/kg drug dose. Similarly, a significant increase in the frequency of MNCE was observed after 12.5 mg/kg HQ treatment at all the post-treatment time periods. The dose effect relationship between various HQ doses and MPCE and MNCE induction was linear and linear quadratic, respectively at all the post-treatment time periods. The PCE/NCE ratio declined in a dose dependent manner at all the post-treatment time periods and this decline was significant when compared to non-drug treated controls. The dose effect relationship was linear quadratic at all the post-treatment time periods studied. © 1997 Elsevier Science Ireland Ltd. Keywords: Hydroquinone; Mouse; Bone marrow; Micronuclei; PCE/NCE ratio
1. Introduction In man many cancers result from environmental carcinogens (Boyland et al., 1967; Higginson et al., 1969) in addition to occupational hazard re-
* Corresponding author. Tel.: + 91 8252 712007/71219, ext.: 2122; fax: + 91 8252 70062.
lated neoplasia. Hydroquinone (HQ) is found in both free and conjugated forms in plants and animals (Ho¨gl et al., 1958; Eisner et al., 1958; Howard et al., 1979; Iguchi et al., 1990). Industrial production also adds to the environmental burden of HQ. HQ is widely used as an antioxidant or stabilizer for certain materials that polymerize in the presence of free radicals, and as a chemical intermediate for the production of anti-
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oxidants, antiozonants, agrochemicals and polymers. It is widely used as a photographic developer for black and white photographs and also in cosmetics and certain medical preparations. Human exposure to HQ may be due to the use of cosmetics and certain medical preparations, plant and animal derived products as well as from cigarette smokes. HQ is found in varying amounts from 100 to 300 mg per cigarette in non-filter cigarettes (IARC, 1986). Therefore, not only the smokers but also the non-smokers inhaling the cigarette smoke are inadvertently exposed to HQ. HQ is also distributed in plant derived food products (wheat germ), in brewed coffee and teas prepared from the leaves of some berries, where the concentration is greater than 1% (Deichmann et al., 1981). A single cup of coffee contains 100 mg of HQ (Gold et al., 1992). Food packaging adhesives also contain HQ (FDA, 1981, 1991). HQ is a benzene metabolite and is implicated in the development of aplastic anemia, leukemia and other blood related disorders (Glodstein et al., 1977; IARC, 1982). HQ has been reported to induce micronuclei in vitro (Glatt et al., 1989, 1990; Antoccia et al., 1991) as well as in vivo (Miller et al., 1989; Xu et al., 1990). HQ treatment of pregnant rabbits and rats has been reported to inhibit the weight gain in dams (Murphy et al., 1992; Krasavage et al., 1992). The detailed studies regarding the effect of low doses of HQ on micronuclei induction and cell proliferation at different post-treatment time periods are lacking. Therefore, the present study was undertaken to obtain an insight into the effects of various doses of HQ in mouse bone marrow on micronuclei-induction at different post-treatment time periods, in order to determine (1) the level of no effect (NOEL) dose of HQ and (2) the time kinetics of micronuclei induction.
2. Material and methods Male Swiss albino mice, 6 – 8 weeks old weighing 25–30 g were selected from an inbred colony maintained under standard conditions.
2.1. Preparation of drug HQ Cat. No. H-9003 (Sigma Chemical, St. Louis, MO) was dissolved in sterile double distilled water (DDW).
2.2. Mode of administration A quantity of 0.01 ml/g body wt of DDW or HQ solution was administered intraperitoneally.
2.3. Experimental protocol The animals were divided into two groups as follows: DDW treated group: the animals of this group received 0.01 ml/g body wt. of DDW. HQ treated group: The animals of this group were injected with 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt. of HQ solution. The animals from both groups were killed at 12, 24 and 36 h after DDW or HQ treatment. The micronuclei were prepared according to the method of Schmid et al. (1975) with certain modifications described by Jagetia et al. (1992). Briefly, the femurs of each animal were dissected out and the bone marrow was flushed out into Dulbecco’s modified Eagle’s medium (DMEM) separately. The suspension was centrifuged. A few drops of fetal calf serum (FCS) were added and the pellet was mixed thoroughly. Smears were drawn on to precleaned coded slides using a drop of the resultant suspension in FCS. The slides were air dried and fixed in absolute methanol. The slides were stained with 0.125% acridine orange (BDH, England, Gurr Cat. No. 34001 9704640E) in Sorensen’s buffer (pH 6.8). The slides were washed twice in Sorensen’s buffer. The slides mounted in Sorensen’s buffer were observed under a fluorescent microscope (Carl Zeiss Photomicroscope III, Germany) using a 40× Neofluar objective. A minimum of 2000 polychromatic erythrocytes (PCE) and 2000 normochromatic erythrocytes (NCE) were counted for the presence of micronuclei (MPCE and MNCE) for each animal. A total of not less than 10 000 either PCE or NCE were counted for each dose of HQ. Data regarding the polychromatic and normochromatic ery-
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throcyte ratio (P/N ratio) was also collected, where a minimum of 4000 erythrocytes per animal was scored. For each drug dose of HQ at each post-treatment time period studied, five animals were used. The statistical significance between control and HQ groups was calculated using Wilcoxon’s signed rank test. The data were fitted on linear (Y =a +ßD) or linear quadratic (Y= C + aD + aD 2) equations to describe the dose response, if any, where C is control micronuclei frequency, D is dose of HQ and a and b are the constants.
3. Results The results are expressed as the frequency of micronuclei (MPCE or MNCE9SEM) per 1000 and PCE/NCE ratio9 SEM in Table 1. The administration of 0.78 – 3.125 mg/kg HQ did not increase the frequency of MPCE significantly at 12 h post-administration. However, a non significant elevation in the MPCE frequency was observed after 3.125 mg/kg HQ at this time period. With the further increase in HQ dose, the frequency of MPCE increased significantly in a dose dependent manner (Fig. 1a). The frequency of MPCE was higher at 24 h post-treatment compared to 12 and 36 h, respectively. This increase in the frequency of MPCE was significant at all the drug doses when compared to non-drug treated controls, except at the lowest dose (0.78 mg/kg) used, where it was within the range of spontaneous level. However a non-significant elevation in the frequency of MPCE was observed after 1.56 mg/kg HQ, when compared to non-drug treated controls. The administration of 6.25 mg/ kg HQ resulted in a two-fold increase in the frequency of MPCE and an increase of HQ dose to 100 mg/kg resulted in 8-fold elevation in the frequency of MPCE when compared to DDW treated control at 24 h post-treatment. Further, the administration of 75 and 100 mg/kg HQ resulted in the induction of MPCE bearing two micronuclei (0.919 0.19 and 1.4 9 0.19, respectively). The frequency of MPCE declined at 36 h post-treatment, however a significant and dose dependent elevation in the frequency of MPCE
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was observed in the bone marrow of mice treated with 6.25 mg/kg HQ treatment. The MPCE for all the post-treatment time periods were fitted on linear and linear quadratic models, where the r values were similar for both models. Therefore, the best fit was considered for a linear model (r= 0.99, 0.98 and 0.99 for 12, 24 and 36 h, respectively). The frequency of MNCE also increased in a dose dependent manner (Fig. 1b) and significant increases were observed after administration of 6.25 and 12.5 mg/kg drug dose at 12, 24 and 36 h post-treatment, respectively. An almost plateau was observed in MNCE frequency after 50 mg/kg of HQ treatment (Fig. 1b). The dose effect relationship was linear quadratic (Table 1). The frequency of MNCE also reached a peak level at 24 h post-treatment and declined thereafter. To ascertain the changes in the cell proliferation, the PCE/NCE ratio was also evaluated. Administration of HQ resulted in a dose dependent inhibition in the erythropoiesis as is evidenced by a continuous decline in the PCE/NCE ratio with increasing drug dose at all the post-treatment time periods studied (Fig. 1c). This decline in the PCE/ NCE ratio was significant with respect to nondrug treated control at all the post treatment time periods (Table 1). The PCE/NCE ratio was lowest at 36 h post-treatment when compared to the other two scoring time periods. The dose response relationship for the PCE/NCE ratio was linear quadratic (r= 0.9, 0.93 and 0.9 for 12, 24 and 36 h, respectively).
4. Discussion In the present study an attempt has been made to assess the clastogenic potential of various doses of HQ in the bone marrow of mice and to find out the dose of HQ which will not elevate the frequency of micronuclei above the spontaneous level. From our results it is clear that HQ administration increased the frequency of micronuclei at all the time periods with a few exceptions. The frequency of MPCE was significantly higher at 24 h post-treatment, except at the lowest dose. How-
0.75 9 0.13 0.529 0.004 0.60 9 0.10 0.789 0.12 1.25 9 0.13 1.67 9 0.12a 2.06 9 0.11a 2.76 9 0.11a 2.86 9 0.15a 3.23 9 0.16a 0.99 0.06 90.008 −0.0004 9 0.0001
0.64 90.11 0.50 90.01 0.59 9 0.10 0.66 9 0.11 1.11 90.11a 1.52 9 0.11a 1.88 90.10a 2.40 9 0.15a 2.58 9 0.09a 2.76 90.12a 0.98 0.05 90.007 −0.0003 9 0.00007
2.89 0.12 2.29 0.12 2.9 9 0.19 3.5 9 0.16 4.19 0.10a 5.39 0.25a 6.79 0.25a 8.69 0.19a 11.89 0.12a 15.49 0.19a 0.99 3.269 0.23 0.129 0.005
2.89 0.12 2.5 9 0.16 3.59 0.16 4.390.12a 5.690.19a 6.690.19a 7.990.19a 9.390.25a 16.29 0.64a 22.19 0.71a 0.98 3.489 0.71 0.7190.01
2.790.12 2.1 90.10 2.6 90.10 3.2 90.12 3.6 90.10a 4.9 90.19a 6.2 90.25a 8.0 90.22a 9.10 9 0.19a 11.9 9 0.19a 0.99 3.281 90.28 0.087 90.006 9 0.00005
0.64 9 0.11 0.49 9 0.01 0.56 9 0.09 0.58 90.09 0.72 90.11 1.02 9 0.10a 1.39 9 0.10a 1.91 9 0.14a 1.93 9 0.12a 2.26 9 0.13a 0.996 0.03 9 0.005 −0.0002
36
24
12
36
12
24
Frequency of MNCE 9 S.E.M. (h)
Frequency of MPCE 9 S.E.M. (h)
p= aB0.03. No symbols= non-significant.
0 0.78 1.56 3.125 6.25 12.5 25 50 75 100 r a b
HQ dose (mg/kg)
9 0.00002
1.079 0.002 1.009 0.01a 0.9890.003a 0.959 0.01a 0.929 0.002a 0.8990.002a 0.8690.006a 0.8390.002a 0.8190.002a 0.7990.002a 0.97 0.0059 0.002 0.00003
12
1.07 9 0.002 1.0390.008a 1.0190.005a 0.9890.008a 0.9590.02a 0.9290.002a 0.8990.002a 0.8690.002a 0.8490.002a 0.8290.002a 0.94 −0.0059 0.001 0.00003 9 0.0001
24
PCE/NCE ratio 9 S.E.M. (h)
1.06 90.004 0.9 99 0.02a 0.9 69 0.006a 0.92 9 0.008a 0.8 99 0.002a 0.85 90.006a 0.82 90.006a 0.79 90.003a 0.77 90.002a 0.75 90.004a 0.90 −0.00690.002 0.0000490.00002
36
Table 1 Effect of different doses of HQ on the micronuclei formation and PCE/NCE ratio in the mouse bone marrow at various post-treatment time periods
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Fig. 1. Effect of various doses of HQ on the induction of micronuclei (a) in PCE (b) in NCE and (c) PCE/NCE ratio in mouse bone marrow.
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ever, a non-significant increase in the frequency of MPCE was observed after 1.56 mg/kg HQ treatment. A two- and eight-fold increase in the frequency of MPCE was observed at 24 h post-treatment in mice treated with 6.25 and 100 mg/kg HQ, respectively. Similarly, HQ has been reported to increase the frequency of micronuclei in C57Bl/Cne ×C3HCne F1) mice at 18 and 24 h post-treatment after administration of 40, 80 and 120 mg/kg body wt. of HQ with a highest elevation at 24 h post-treatment, where this increase was significant (Pacchierotti et al., 1991). The frequency of micronuclei has been found to be elevated in mice treated with 50, 75 and 100 mg/kg HQ treatment (Adler et al., 1990). An increase in the micronuclei frequency in mouse bone marrow after intraperitoneal administration of HQ has been reported before (Gocke et al., 1981; Ciranni et al., 1988a,b; Barale et al., 1990). Chronic exposure of mice to 25 – 100 mg/kg HQ has been found to increase the frequency of micronuclei in mouse bone marrow (Tunek et al., 1982). HQ has been reported to induced micronuclei in V79 cells in vitro (Glatt et al., 1989, 1990), rat intestinal cells and human liver cells (Glatt et al., 1990) in cultured human lymphocytes (Yager et al., 1990; Robertson et al., 1991; Bigatti et al., 1994) and Chinese hamster embryonic lung cells (Antoccia et al., 1991). However, in the present study we have consistently observed a significant increase in the frequency of micronuclei at all the three post-injection scoring time periods at all the doses studied, except 0.78 and 1.56 mg/kg. In the present study we have separated the frequency of micronuclei into MPCE and MNCE and still observed a significant elevation (Table 1). The frequency of micronuclei increased in a dose dependent manner at all the post-treatment time periods studied and the dose effect relationship for MPCE was linear, while it was linear quadratic for MNCE. This is at variance with the non-linear dose response reported in mice treated with 30, 50, 75 and 100 mg/kg HQ (Adler et al., 1990). We have observed a peak level of micronuclei at 24 h post-treatment, which is in conformation with the earlier findings, where a peak frequency of micronuclei was observed at 24 h post-treatment (Adler et al., 1990; Pacchierotti et
al., 1991). The time kinetics of micronuclei-induction has been studied before (Adler et al., 1990), however, the time periods chosen in the present study are different than that of the earlier study (Adler et al., 1990), except 24 h post-treatment. The frequency of MNCE reached a plateau phase after administration of 50 mg/kg HQ, indicating its toxic effects above this dose. The observation of micronuclei at low doses may be owing to the variation in species sensitivity to HQ treatment. Since HQ has been reported to exhibit variation in sensitivity in different species. Cats have been reported to be the most sensitive species (LD50 40–85 mg/kg) (IPCS, 1994). HQ has also been reported to increase the frequency of structural chromosome aberrations significantly after 100 mg/kg body wt. in (101/ E1× C3H/E1) F1 mice from 6 to 24 h post-treatment. This effect was observable at 24 h even at 75 mg/kg dose (Xu et al., 1990). Similarly, HQ has been reported to increase the frequency of chromosomal aberrations in mice sperm (Ciranni et al., 1991). The HQ administration resulted in a significant and dose dependent decline in the cell proliferation as evidenced by the continuous decline in the PCE/NCE ratio at all the post-treatment time periods. Even the lowest dose of 0.78 mg/kg also reduced the PCE/NCE ratio significantly at all the three post-treatment time periods. This decline in the PCE/NCE ratio is an indication of bone marrow toxicity of HQ owing to the impairment of spindle apparatus. The lowest PCE/NCE ratio was observed at 36 h post-treatment. A decline in the PCE/NCE ratio has been reported in the mice treated with various doses of HQ (Adler et al., 1990). HQ has been reported to cause an abnormal microtubule assembly at a concentration of 110 mg/l (Wallin et al., 1993). It has also been reported to inhibit the microtubule assembly in a concentration-dependent manner (Irons et al., 1981) and the oxidation product of HQ has been reported to inhibit tubulin assembly even at low concentrations (Epe et al., 1990). The inhibition of microtubule assembly by HQ may be responsible for the dose dependent decline in the PCE/ NCE ratio.
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HQ absorption through oral and intratracheal routes is rapid and extensive (Garton et al., 1949; Divincenzo et al., 1984; English et al., 1988) and it is mainly converted to p-benzoquinone (0.25 – 7%) and or HQ-monoglucuronide and HQ monosulphate (\90%) (Divincenzo et al., 1984; English et al., 1988). The two major toxic metabolites of HQ are p-benzoquinone and p-benzosemiquinone, which may also be responsible for the observed clastogenic effect apart from the HQ it self. The HQ administration has been reported to induce single strand breaks of DNA in hepatocytes (Walles et al., 1992) and chromosomal aberrations in Chinese hamster ovary cells (Galloway et al., 1987). The ability of HQ to induce DNA damage and chromosome aberrations may be responsible for the production of micronuclei, which are formed from acentric fragments or whole chromosome that lag behind during cell division (Heddle et al., 1977; Yamamoto et al., 1980). HQ produces micronuclei by clastogenesis and spindle impairment (Irons et al., 1981), which may also be one of the reasons of micronuclei-induction even at low doses. The doses selected (especially low doses) in the present investigation fall in the dose ranges to which humans are inadvertently exposed. The human exposure to HQ is mainly through the consumption of food materials containing varying amounts of HQ viz. wheat germ (8.352 mg/kg), whole wheat (0.893 – 0.992 mg/kg), pear skin (38.057 mg/kg) and pear flesh (1.301 mg/kg) (IPCS, 1994) and coffee and tea (\ 1%) (Deichmann et al., 1981). The other sources of human exposures are smoking, where each cigarette contains 100–300 mg of HQ (IARC, 1986) and use of cosmetics, which contains 1.5 – 2% HQ or as much as 7% (Brauer et al., 1985; Godlee et al., 1992). The occupational exposure is mainly during the manufacture of HQ and also during development of black and white photographs. Deaths have been reported after accidental ingestion of photographic developer in the dose range of 80 – 300 mg/kg body wt. (Busatto et al., 1939; Zeidman et al., 1945). It is clear from the above that various authors, who have investigated the effect of HQ did not use HQ doses below 25 mg/kg. We have evaluated
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the effect of HQ up to a no effect level (NOEL) dose, which is 0.78 mg/kg, where the frequencies of micronuclei are within the spontaneous range. However, a further increase in the HQ dose to 1.56 mg/kg resulted in a non significant increase in the frequency of micronuclei. This increase implies a low level of clastogenicity by HQ.
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Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow G.C. Jagetia *, R. Aruna Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India Received 24 June 1997; received in revised form 6 October 1997; accepted 6 October 1997
Abstract The frequency of micronuclei (micronucleated polychromatic erythrocytes, MPCE and micronucleated normochromatic erythrocytes, MNCE) was studied at 12, 24 and 36 h post-treatment in the bone marrow of mice treated with 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt of hydroquinone (HQ). Treatment of mice with various doses of HQ resulted in a dose dependent increase in the frequency of both MPCE and MNCE at all the post-treatment time periods. The frequency of MPCE was significantly higher after administration of 3.125 mg/kg HQ at 24 h post-treatment, except 12 and 36 h, where a significant increase in the frequency of MPCE was observed only after administration of 6.25 mg/kg drug dose. Similarly, a significant increase in the frequency of MNCE was observed after 12.5 mg/kg HQ treatment at all the post-treatment time periods. The dose effect relationship between various HQ doses and MPCE and MNCE induction was linear and linear quadratic, respectively at all the post-treatment time periods. The PCE/NCE ratio declined in a dose dependent manner at all the post-treatment time periods and this decline was significant when compared to non-drug treated controls. The dose effect relationship was linear quadratic at all the post-treatment time periods studied. © 1997 Elsevier Science Ireland Ltd. Keywords: Hydroquinone; Mouse; Bone marrow; Micronuclei; PCE/NCE ratio
1. Introduction In man many cancers result from environmental carcinogens (Boyland et al., 1967; Higginson et al., 1969) in addition to occupational hazard re-
* Corresponding author. Tel.: + 91 8252 712007/71219, ext.: 2122; fax: + 91 8252 70062.
lated neoplasia. Hydroquinone (HQ) is found in both free and conjugated forms in plants and animals (Ho¨gl et al., 1958; Eisner et al., 1958; Howard et al., 1979; Iguchi et al., 1990). Industrial production also adds to the environmental burden of HQ. HQ is widely used as an antioxidant or stabilizer for certain materials that polymerize in the presence of free radicals, and as a chemical intermediate for the production of anti-
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oxidants, antiozonants, agrochemicals and polymers. It is widely used as a photographic developer for black and white photographs and also in cosmetics and certain medical preparations. Human exposure to HQ may be due to the use of cosmetics and certain medical preparations, plant and animal derived products as well as from cigarette smokes. HQ is found in varying amounts from 100 to 300 mg per cigarette in non-filter cigarettes (IARC, 1986). Therefore, not only the smokers but also the non-smokers inhaling the cigarette smoke are inadvertently exposed to HQ. HQ is also distributed in plant derived food products (wheat germ), in brewed coffee and teas prepared from the leaves of some berries, where the concentration is greater than 1% (Deichmann et al., 1981). A single cup of coffee contains 100 mg of HQ (Gold et al., 1992). Food packaging adhesives also contain HQ (FDA, 1981, 1991). HQ is a benzene metabolite and is implicated in the development of aplastic anemia, leukemia and other blood related disorders (Glodstein et al., 1977; IARC, 1982). HQ has been reported to induce micronuclei in vitro (Glatt et al., 1989, 1990; Antoccia et al., 1991) as well as in vivo (Miller et al., 1989; Xu et al., 1990). HQ treatment of pregnant rabbits and rats has been reported to inhibit the weight gain in dams (Murphy et al., 1992; Krasavage et al., 1992). The detailed studies regarding the effect of low doses of HQ on micronuclei induction and cell proliferation at different post-treatment time periods are lacking. Therefore, the present study was undertaken to obtain an insight into the effects of various doses of HQ in mouse bone marrow on micronuclei-induction at different post-treatment time periods, in order to determine (1) the level of no effect (NOEL) dose of HQ and (2) the time kinetics of micronuclei induction.
2. Material and methods Male Swiss albino mice, 6 – 8 weeks old weighing 25–30 g were selected from an inbred colony maintained under standard conditions.
2.1. Preparation of drug HQ Cat. No. H-9003 (Sigma Chemical, St. Louis, MO) was dissolved in sterile double distilled water (DDW).
2.2. Mode of administration A quantity of 0.01 ml/g body wt of DDW or HQ solution was administered intraperitoneally.
2.3. Experimental protocol The animals were divided into two groups as follows: DDW treated group: the animals of this group received 0.01 ml/g body wt. of DDW. HQ treated group: The animals of this group were injected with 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt. of HQ solution. The animals from both groups were killed at 12, 24 and 36 h after DDW or HQ treatment. The micronuclei were prepared according to the method of Schmid et al. (1975) with certain modifications described by Jagetia et al. (1992). Briefly, the femurs of each animal were dissected out and the bone marrow was flushed out into Dulbecco’s modified Eagle’s medium (DMEM) separately. The suspension was centrifuged. A few drops of fetal calf serum (FCS) were added and the pellet was mixed thoroughly. Smears were drawn on to precleaned coded slides using a drop of the resultant suspension in FCS. The slides were air dried and fixed in absolute methanol. The slides were stained with 0.125% acridine orange (BDH, England, Gurr Cat. No. 34001 9704640E) in Sorensen’s buffer (pH 6.8). The slides were washed twice in Sorensen’s buffer. The slides mounted in Sorensen’s buffer were observed under a fluorescent microscope (Carl Zeiss Photomicroscope III, Germany) using a 40× Neofluar objective. A minimum of 2000 polychromatic erythrocytes (PCE) and 2000 normochromatic erythrocytes (NCE) were counted for the presence of micronuclei (MPCE and MNCE) for each animal. A total of not less than 10 000 either PCE or NCE were counted for each dose of HQ. Data regarding the polychromatic and normochromatic ery-
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throcyte ratio (P/N ratio) was also collected, where a minimum of 4000 erythrocytes per animal was scored. For each drug dose of HQ at each post-treatment time period studied, five animals were used. The statistical significance between control and HQ groups was calculated using Wilcoxon’s signed rank test. The data were fitted on linear (Y =a +ßD) or linear quadratic (Y= C + aD + aD 2) equations to describe the dose response, if any, where C is control micronuclei frequency, D is dose of HQ and a and b are the constants.
3. Results The results are expressed as the frequency of micronuclei (MPCE or MNCE9SEM) per 1000 and PCE/NCE ratio9 SEM in Table 1. The administration of 0.78 – 3.125 mg/kg HQ did not increase the frequency of MPCE significantly at 12 h post-administration. However, a non significant elevation in the MPCE frequency was observed after 3.125 mg/kg HQ at this time period. With the further increase in HQ dose, the frequency of MPCE increased significantly in a dose dependent manner (Fig. 1a). The frequency of MPCE was higher at 24 h post-treatment compared to 12 and 36 h, respectively. This increase in the frequency of MPCE was significant at all the drug doses when compared to non-drug treated controls, except at the lowest dose (0.78 mg/kg) used, where it was within the range of spontaneous level. However a non-significant elevation in the frequency of MPCE was observed after 1.56 mg/kg HQ, when compared to non-drug treated controls. The administration of 6.25 mg/ kg HQ resulted in a two-fold increase in the frequency of MPCE and an increase of HQ dose to 100 mg/kg resulted in 8-fold elevation in the frequency of MPCE when compared to DDW treated control at 24 h post-treatment. Further, the administration of 75 and 100 mg/kg HQ resulted in the induction of MPCE bearing two micronuclei (0.919 0.19 and 1.4 9 0.19, respectively). The frequency of MPCE declined at 36 h post-treatment, however a significant and dose dependent elevation in the frequency of MPCE
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was observed in the bone marrow of mice treated with 6.25 mg/kg HQ treatment. The MPCE for all the post-treatment time periods were fitted on linear and linear quadratic models, where the r values were similar for both models. Therefore, the best fit was considered for a linear model (r= 0.99, 0.98 and 0.99 for 12, 24 and 36 h, respectively). The frequency of MNCE also increased in a dose dependent manner (Fig. 1b) and significant increases were observed after administration of 6.25 and 12.5 mg/kg drug dose at 12, 24 and 36 h post-treatment, respectively. An almost plateau was observed in MNCE frequency after 50 mg/kg of HQ treatment (Fig. 1b). The dose effect relationship was linear quadratic (Table 1). The frequency of MNCE also reached a peak level at 24 h post-treatment and declined thereafter. To ascertain the changes in the cell proliferation, the PCE/NCE ratio was also evaluated. Administration of HQ resulted in a dose dependent inhibition in the erythropoiesis as is evidenced by a continuous decline in the PCE/NCE ratio with increasing drug dose at all the post-treatment time periods studied (Fig. 1c). This decline in the PCE/ NCE ratio was significant with respect to nondrug treated control at all the post treatment time periods (Table 1). The PCE/NCE ratio was lowest at 36 h post-treatment when compared to the other two scoring time periods. The dose response relationship for the PCE/NCE ratio was linear quadratic (r= 0.9, 0.93 and 0.9 for 12, 24 and 36 h, respectively).
4. Discussion In the present study an attempt has been made to assess the clastogenic potential of various doses of HQ in the bone marrow of mice and to find out the dose of HQ which will not elevate the frequency of micronuclei above the spontaneous level. From our results it is clear that HQ administration increased the frequency of micronuclei at all the time periods with a few exceptions. The frequency of MPCE was significantly higher at 24 h post-treatment, except at the lowest dose. How-
0.75 9 0.13 0.529 0.004 0.60 9 0.10 0.789 0.12 1.25 9 0.13 1.67 9 0.12a 2.06 9 0.11a 2.76 9 0.11a 2.86 9 0.15a 3.23 9 0.16a 0.99 0.06 90.008 −0.0004 9 0.0001
0.64 90.11 0.50 90.01 0.59 9 0.10 0.66 9 0.11 1.11 90.11a 1.52 9 0.11a 1.88 90.10a 2.40 9 0.15a 2.58 9 0.09a 2.76 90.12a 0.98 0.05 90.007 −0.0003 9 0.00007
2.89 0.12 2.29 0.12 2.9 9 0.19 3.5 9 0.16 4.19 0.10a 5.39 0.25a 6.79 0.25a 8.69 0.19a 11.89 0.12a 15.49 0.19a 0.99 3.269 0.23 0.129 0.005
2.89 0.12 2.5 9 0.16 3.59 0.16 4.390.12a 5.690.19a 6.690.19a 7.990.19a 9.390.25a 16.29 0.64a 22.19 0.71a 0.98 3.489 0.71 0.7190.01
2.790.12 2.1 90.10 2.6 90.10 3.2 90.12 3.6 90.10a 4.9 90.19a 6.2 90.25a 8.0 90.22a 9.10 9 0.19a 11.9 9 0.19a 0.99 3.281 90.28 0.087 90.006 9 0.00005
0.64 9 0.11 0.49 9 0.01 0.56 9 0.09 0.58 90.09 0.72 90.11 1.02 9 0.10a 1.39 9 0.10a 1.91 9 0.14a 1.93 9 0.12a 2.26 9 0.13a 0.996 0.03 9 0.005 −0.0002
36
24
12
36
12
24
Frequency of MNCE 9 S.E.M. (h)
Frequency of MPCE 9 S.E.M. (h)
p= aB0.03. No symbols= non-significant.
0 0.78 1.56 3.125 6.25 12.5 25 50 75 100 r a b
HQ dose (mg/kg)
9 0.00002
1.079 0.002 1.009 0.01a 0.9890.003a 0.959 0.01a 0.929 0.002a 0.8990.002a 0.8690.006a 0.8390.002a 0.8190.002a 0.7990.002a 0.97 0.0059 0.002 0.00003
12
1.07 9 0.002 1.0390.008a 1.0190.005a 0.9890.008a 0.9590.02a 0.9290.002a 0.8990.002a 0.8690.002a 0.8490.002a 0.8290.002a 0.94 −0.0059 0.001 0.00003 9 0.0001
24
PCE/NCE ratio 9 S.E.M. (h)
1.06 90.004 0.9 99 0.02a 0.9 69 0.006a 0.92 9 0.008a 0.8 99 0.002a 0.85 90.006a 0.82 90.006a 0.79 90.003a 0.77 90.002a 0.75 90.004a 0.90 −0.00690.002 0.0000490.00002
36
Table 1 Effect of different doses of HQ on the micronuclei formation and PCE/NCE ratio in the mouse bone marrow at various post-treatment time periods
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Fig. 1. Effect of various doses of HQ on the induction of micronuclei (a) in PCE (b) in NCE and (c) PCE/NCE ratio in mouse bone marrow.
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ever, a non-significant increase in the frequency of MPCE was observed after 1.56 mg/kg HQ treatment. A two- and eight-fold increase in the frequency of MPCE was observed at 24 h post-treatment in mice treated with 6.25 and 100 mg/kg HQ, respectively. Similarly, HQ has been reported to increase the frequency of micronuclei in C57Bl/Cne ×C3HCne F1) mice at 18 and 24 h post-treatment after administration of 40, 80 and 120 mg/kg body wt. of HQ with a highest elevation at 24 h post-treatment, where this increase was significant (Pacchierotti et al., 1991). The frequency of micronuclei has been found to be elevated in mice treated with 50, 75 and 100 mg/kg HQ treatment (Adler et al., 1990). An increase in the micronuclei frequency in mouse bone marrow after intraperitoneal administration of HQ has been reported before (Gocke et al., 1981; Ciranni et al., 1988a,b; Barale et al., 1990). Chronic exposure of mice to 25 – 100 mg/kg HQ has been found to increase the frequency of micronuclei in mouse bone marrow (Tunek et al., 1982). HQ has been reported to induced micronuclei in V79 cells in vitro (Glatt et al., 1989, 1990), rat intestinal cells and human liver cells (Glatt et al., 1990) in cultured human lymphocytes (Yager et al., 1990; Robertson et al., 1991; Bigatti et al., 1994) and Chinese hamster embryonic lung cells (Antoccia et al., 1991). However, in the present study we have consistently observed a significant increase in the frequency of micronuclei at all the three post-injection scoring time periods at all the doses studied, except 0.78 and 1.56 mg/kg. In the present study we have separated the frequency of micronuclei into MPCE and MNCE and still observed a significant elevation (Table 1). The frequency of micronuclei increased in a dose dependent manner at all the post-treatment time periods studied and the dose effect relationship for MPCE was linear, while it was linear quadratic for MNCE. This is at variance with the non-linear dose response reported in mice treated with 30, 50, 75 and 100 mg/kg HQ (Adler et al., 1990). We have observed a peak level of micronuclei at 24 h post-treatment, which is in conformation with the earlier findings, where a peak frequency of micronuclei was observed at 24 h post-treatment (Adler et al., 1990; Pacchierotti et
al., 1991). The time kinetics of micronuclei-induction has been studied before (Adler et al., 1990), however, the time periods chosen in the present study are different than that of the earlier study (Adler et al., 1990), except 24 h post-treatment. The frequency of MNCE reached a plateau phase after administration of 50 mg/kg HQ, indicating its toxic effects above this dose. The observation of micronuclei at low doses may be owing to the variation in species sensitivity to HQ treatment. Since HQ has been reported to exhibit variation in sensitivity in different species. Cats have been reported to be the most sensitive species (LD50 40–85 mg/kg) (IPCS, 1994). HQ has also been reported to increase the frequency of structural chromosome aberrations significantly after 100 mg/kg body wt. in (101/ E1× C3H/E1) F1 mice from 6 to 24 h post-treatment. This effect was observable at 24 h even at 75 mg/kg dose (Xu et al., 1990). Similarly, HQ has been reported to increase the frequency of chromosomal aberrations in mice sperm (Ciranni et al., 1991). The HQ administration resulted in a significant and dose dependent decline in the cell proliferation as evidenced by the continuous decline in the PCE/NCE ratio at all the post-treatment time periods. Even the lowest dose of 0.78 mg/kg also reduced the PCE/NCE ratio significantly at all the three post-treatment time periods. This decline in the PCE/NCE ratio is an indication of bone marrow toxicity of HQ owing to the impairment of spindle apparatus. The lowest PCE/NCE ratio was observed at 36 h post-treatment. A decline in the PCE/NCE ratio has been reported in the mice treated with various doses of HQ (Adler et al., 1990). HQ has been reported to cause an abnormal microtubule assembly at a concentration of 110 mg/l (Wallin et al., 1993). It has also been reported to inhibit the microtubule assembly in a concentration-dependent manner (Irons et al., 1981) and the oxidation product of HQ has been reported to inhibit tubulin assembly even at low concentrations (Epe et al., 1990). The inhibition of microtubule assembly by HQ may be responsible for the dose dependent decline in the PCE/ NCE ratio.
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HQ absorption through oral and intratracheal routes is rapid and extensive (Garton et al., 1949; Divincenzo et al., 1984; English et al., 1988) and it is mainly converted to p-benzoquinone (0.25 – 7%) and or HQ-monoglucuronide and HQ monosulphate (\90%) (Divincenzo et al., 1984; English et al., 1988). The two major toxic metabolites of HQ are p-benzoquinone and p-benzosemiquinone, which may also be responsible for the observed clastogenic effect apart from the HQ it self. The HQ administration has been reported to induce single strand breaks of DNA in hepatocytes (Walles et al., 1992) and chromosomal aberrations in Chinese hamster ovary cells (Galloway et al., 1987). The ability of HQ to induce DNA damage and chromosome aberrations may be responsible for the production of micronuclei, which are formed from acentric fragments or whole chromosome that lag behind during cell division (Heddle et al., 1977; Yamamoto et al., 1980). HQ produces micronuclei by clastogenesis and spindle impairment (Irons et al., 1981), which may also be one of the reasons of micronuclei-induction even at low doses. The doses selected (especially low doses) in the present investigation fall in the dose ranges to which humans are inadvertently exposed. The human exposure to HQ is mainly through the consumption of food materials containing varying amounts of HQ viz. wheat germ (8.352 mg/kg), whole wheat (0.893 – 0.992 mg/kg), pear skin (38.057 mg/kg) and pear flesh (1.301 mg/kg) (IPCS, 1994) and coffee and tea (\ 1%) (Deichmann et al., 1981). The other sources of human exposures are smoking, where each cigarette contains 100–300 mg of HQ (IARC, 1986) and use of cosmetics, which contains 1.5 – 2% HQ or as much as 7% (Brauer et al., 1985; Godlee et al., 1992). The occupational exposure is mainly during the manufacture of HQ and also during development of black and white photographs. Deaths have been reported after accidental ingestion of photographic developer in the dose range of 80 – 300 mg/kg body wt. (Busatto et al., 1939; Zeidman et al., 1945). It is clear from the above that various authors, who have investigated the effect of HQ did not use HQ doses below 25 mg/kg. We have evaluated
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the effect of HQ up to a no effect level (NOEL) dose, which is 0.78 mg/kg, where the frequencies of micronuclei are within the spontaneous range. However, a further increase in the HQ dose to 1.56 mg/kg resulted in a non significant increase in the frequency of micronuclei. This increase implies a low level of clastogenicity by HQ.
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Cancer Res. 50, 393 – 399. Yamamoto, K.I., Kikuchi, Y., 1980. A comparison of diameters of micronuclei-induced by clastogens and spindle poisons. Mutat. Res. 71, 127 – 131. Zeidman, I., Deutl, R., 1945. Poisoning by hydroquinone and mono-methyl-paraaminophenol sulfate. Am. J. Med. Sci. 210, 328 – 333.