Transplacental inhibitory effect of carrot juice on the clastogenicity of cyclophosphamide in mice

Transplacental inhibitory effect of carrot juice on the clastogenicity of cyclophosphamide in mice

Maria Clara Gimmler-Luz, Valesca Veiga Cardoso, Cassius Ugarte Sardiglia and Débora da Silva Widholzer

Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Caixa Postal 15053, 91501-970 Porto Alegre, RS, Brasil. Send correspondence to M.C.G.-L., Travessa da Saúde, 65, Apto. 303, 90540-090 Porto Alegre, RS. Fax:[phone].

ABSTRACT

Genetic damage during the prenatal period can provoke important neoplastic alterations and other diseases in postnatal life. Beta-carotene (ßC) is considered to be one of the most important anticarcinogens in the diet and can protect mammalian cells against genotoxic events. As carrots are important dietary source of ßC, we decided to test the effect of fresh carrot juice (CaJ) on cyclophosphamide (CP)-induced genotoxicity in maternal and fetal erythropoietic tissues. The treatment with CaJ started on the 7th day of the pregnancy of BALB/c female mice. We observed, on the 16th gestational day, that this treatment did not modify the spontaneous frequency of micronucleated polychromatic erythrocytes (mPCE) in the bone marrow of the females nor in the livers of their fetuses. The mPCE frequency observed 24 h after an intraperitoneal injection of CP (40 mg/kg) on the 15th day was significantly lower in CaJ-pretreated pregnant female bone marrow and in the liver of their fetuses than those observed in the group treated with CP only. These results demonstrate the presence of natural anticlastogens in carrots.

INTRODUCTION

Consumption of naturally occurring compounds can modify the mutagenic and carcinogenic effects of environmental contaminants. Among these natural compounds are dietary antioxidants, including carotenoids such as beta-carotene (ßC), which has extremely low toxicity and is widely distributed in the plant kingdom. The protective action of ßC against oxidative stress was reviewed by Rousseau et al. (1992).

Intake of food rich in ßC is correlated with reduced risk of cancer and coronary disease mortality (Pandey et al., 1995; Poppel and Goldbohm, 1995; Flagg et al. 1995). ßC supplementation reduces the micronucleus (MN) count in epithelial cells of heavy smokers´ sputum and in lymphocytes of healthy volunteers exposed to X-rays (Poppel et al., 1992; Umegaki et al., 1994a). Nevertheless, no significant correlation was found between plasma ßC and DNA adduct levels in lymphocytes of healthy smoking and nonsmoking men (Wang et al., 1997). Conclusive demonstration of reduction in lung cancer risk by ßC supplementation among middle-aged male smokers could not be found by Buring and Hennekens (1995). Reduction in lung cancer risk (most apparent in squamous cell carcinomas) was more pronounced for intakers of dark green and/or dark yellow-orange vegetables, such as carrots, than for carotenoid intakers (Ziegler et al., 1986). Epidemiological observations thus could not resolve whether cancer prevention is due to ßC or to other components of vegetables that are rich in ßC.

Under experimental conditions, ßC possesses a protective effect against two-stage skin carcinogenesis in mice (Kornhauser et al., 1994), but alpha-carotene shows higher potency than ßC in suppressing tumor promotion in mouse skin and lung carcinogenesis models (Nishino, 1995). It has been suggested that the cancer preventive action of carotenoids is through its effect on gene expression. Bertram and Bortkiewicz (1995) observed that the ability of carotenoids to inhibit neoplastic transformation can be correlated to their up-regulatory effect on the expression of connexin 43, a protein involved in cell communication. Zhang et al. (1996) suggested that a lack of RARß expression, a gene that encodes a nuclear receptor for retinoic acid, may be involved in the development of human lung and breast cancer, and is possibly required for the growth inhibitory effect of retinoids in cancer cells. On the other hand, it was suggested that benzo(a)pirene (BaP) induces cell proliferation through mutations in the tumor-suppresser gene (p53) in hamster tracheal epithelium. In these situations ßC did not affect cell proliferation and p53-protein expression (Wolterbeek et al., 1995).

ßC can inhibit the clastogenic effect of methyl methanesulfonate (MMS), 4-nitroquinoleine-1-oxide and cyclophosphamide (CP) in cultured mammalian cells, but not that of some phenolic acids, of H₂O₂ or of mitomycin C (MMC) (Stich and Dunn, 1986; Salvadori et al., 1993). In addition, it can protect cells against 8-methoxypsoralen phototoxicity (Kornhauser et al., 1994). In vivo studies show some contradictory results, perhaps due to the use of different species and administration routes. Gene mutations induced in rat spleen lymphocytes by the carcinogen N-ethyl-N-nitrosourea (ENU) and chromosome damage induced in Chinese hamster bone marrow by MMS, busulfan and thio-TEPA were reduced by ßC, but not the chromosome aberrations produced by CP (Aidoo et al., 1995; Renner, 1985). In mice, ßC protects bone marrow cells against the genotoxic effect of MMC, BaP and CP (Raj and Katz, 1985; Mukherjee et al., 1991; Salvadori et al., 1992). Although a protective effect of ßC against X- and g-radiation was observed in mouse bone marrow cells (Abraham et al., 1993; Umegaki et al., 1994b), Salvadori et al. (1996) found a reduction in X-ray-induced MN after ßC treatment in splenocytes, reticulocytes and spermatides, but not in bone marrow cells. Similarly, despite the protection against DNA single-strand breaks induced by BaP in murine forestomach mucosa, Lahiri et al. (1993) could not find protection of ßC oral pretreatment against MN induced in bone marrow cells. The latter results are interpreted as a restriction of ßC´s protective activity against BaP damage to the target organ.

Genetic damage to fetus may be an important source of neoplasic alterations and other diseases in postnatal life. As carrot is inexpensive, easily available and is a classical source of ßC, we decided to test if fresh carrot juice, a complex mixture, can also protect pregnant females and their fetuses from CP-induced genotoxicity. We chose two tissues to investigate chromosomal damage in erythropoietic cells: adult bone marrow, with the classic MN test described by Schmid (1975), and fetal liver, using the transplacental MN assay developed by Cole et al. (1979). The transplacental assay presents advantages, because CP metabolic activation probably occurs in the liver of the fetus (Harper et al., 1989), close to the target cells (Stoyel and Clark, 1980).

MATERIAL AND METHODS

Inbred BALB/c mice from our own colony, housed with food (mouse cube Nuvilab, CR1, Moinho Nuvipal Ltda., Curitiba, PR) and water ad libitum in the animal house facilities at Instituto de Biociências, Universidade Federal do Rio Grande do Sul, were used in the experiments. One male was placed with three females for mating, and day one of the pregnancy was determined by the presence of a vaginal plug.

Carrots, purchased from a local market, were finely chopped in a food processor. The juice obtained was filtered through gauze, diluted in drinking water at 1:3 (v/v) and named carrot juice (CaJ). ßC can protect mice cells against genotoxicity induced by g-radiation and CP after 7 days of feeding (Mukherjee et al., 1991; Abraham et al., 1993). So, to test carrot action we allowed pregnant females to drink fresh CaJ instead of water, from the 7th gestational day to the end of the experiment. CP induces MN peaks 24 h after a single treatment in maternal and fetal tissues (Harper et al., 1989). Therefore, to evaluate the modulatory action of CaJ on CP-induced clastogenicity, some CaJ-pretreated females received , on the 15th gestational day, a 40 mg/kg intraperitoneal (ip) injection of CP (Enduxan, Abbot Laboratórios do Brasil Ltda., São Paulo, SP), dissolved in distilled water. Controls received water treatments, and the positive control group also received ip CP treatment. The ip administrations were done at a rate of 10 ml/kg body weight. All pregnant females were sacrificed on the 16th gestational day, 24 h after the ip treatment.

Adult bone marrow was collected and prepared as described by Salamone et al. (1980). The liver of each fetus (three per female, removed from the side opposite to maternal ip dosage) was disrupted in fetal bovine serum. The homogeneous preparation was distributed in duplicate coded slides, air-dried, fixed in methanol and stained with May-Grunwald-Giemsa solution. The frequency of polychromatic erythrocytes (PCE) was obtained by counting 500 erythrocytes (PCE plus normochromatic erythrocytes) per slide, 1000 per animal. Micronucleated PCE (mPCE) were scored among 2000 PCE for each individual, 1000 cells in each slide. The results obtained were statistically analyzed by the Mann-Whitney test.

RESULTS AND DISCUSSION

The proportion of PCE was not statistically different from those of the negative control animals in any of the experimental groups, suggesting that CaJ, CP or the combined treatment does not induce erythropoietic cell toxicity (Table I). CaJ did not modify the spontaneous frequency of mPCE in pregnant females nor in their fetuses. CaJ also does not affect point mutations in Salmonella, repairable DNA damage in Escherichia coli, structural chromosome aberrations or sister chromatid exchanges (SCE) in cultured mammalian cell assays (Kassie et al., 1996). In all situations, alone or in combination with CaJ, CP induced a two-times higher mPCE frequency in fetuses than in their mothers. Nevertheless, in CaJ-pretreated animals a significant reduction of the mPCE frequency induced by CP was observed in both, maternal bone marrow and fetal liver. Abraham et al. (1986) also found that a single oral dose of 0.15 ml/kg undiluted carrot juice administered immediately after ip injection of CP reduced the induction of mPCE in adult mice bone marrow. On the other hand, when the mutagen was 7,12-dimethylbenz(a) anthracene, a one-week pretreatment with diluted carrot juice ad libitum did not prevent the induction of chromosome aberrations in rat bone marrow cells (Ito et al., 1986). Darroudi et al. (1988) observed that the frequency of SCE in cultured human lymphocytes and Chinese hamster ovary cells (CHO) was lower when plasma of rats pretreated with carrot juice was added in comparison with plasma of rats treated with CP only. The inhibitory effect was higher in CHO than in human cells which can, at least in part, metabolize CP. No influence of carrot pretreatment was found when the rats received the direct acting metabolite, phosphoramide mustard. For that reason, the authors suggested that carrot juice possibly interferes with enzymatic processes of CP activation.

Table I
- Effect of carrot juice on the spontaneous and cyclophosphamide-induced frequencies of micronucleated polychromatic erythrocytes.

Treatment		Nº of analyzed animals	PCE	mPCE/1000 PCE, Individual data	Mean ± SD	PCE/ PCE + NCE
0	BM	3	6.000	2.5; 3; 2	2.5 ± 0.6	0.55 ± 0.10
	FL	9	18.000	0.5; 2; 1; 1; 0.5; 1.5; 1.5; 0.5; 1	1.0 ± 0.5	0.62 ± 0.05
CaJ	BM	4	8.000	1.5; 2.5; 1.5; 3.5	2.3 ± 1.0	0.43 ± 0.03
	FL	12	24.000	2.5; 2.5; 1; 0.5; 0.5; 0.5; 2.5; 0; 3.5; 2; 1.5; 1	1.5 ± 1.1	0.59 ± 0.08
CP	BM	5	10.000	20.5; 26.5; 22; 30; 31	26.0 ± 4.7	0.51 ± 0.13
	FL	15	30.000	58; 62.5; 49.5; 66; 56; 48; 34; 33.5; 30.5; 59.5; 46; 39.5; 39; 40; 40	43.0 ± 16.0	0.58 ± 0.09
CaJ + CP	BM	9	18.000	19; 16; 13; 14; 16; 16; 22.5; 15.5; 16.5	16.5 ± 2.8*	0.43 ± 0.07
	FL	27	54.000	60.5; 57.5; 53.5; 11.5; 10; 12.5; 9; 20; 17; 47.5; 32; 45; 34; 22; 20; 24; 22; 23.5; 48; 33; 33.5; 21; 24.5; 23; 24.5; 13.5; 29.5	28.6 ± 14.7*	0.54 ± 0.04

0 = Negative control group; CaJ = carrot juice; CP = 40 mg/kg of cyclophosphamide; BM = pregnant female bone marrow; FL = fetal liver; PCE = polychromatic erythrocytes; mPCE = micronucleated PCE; NCE = normochromatic erythrocytes; * = significantly lower than positive control group, treated with CP only (P < 0.001).

From our results (Table I) we conclude that CaJ can protect fetal cells against CP-induced lesions, even if administered to the mother during pregnancy. This suggests that modulation of CP activity by CaJ occurs in the mother and is reflected in the fetus or that modulatory mechanisms also occur in the fetus. Elevated fetal sensitivity and the fact that the embryotoxicity of CP depends more on the fetal than the maternal genotype were discussed by Harper et al. (1989), who suggested a direct fetal liver CP activation. Comparison of our results with those of literature reveals some common points. If the reduction in frequency observed by us in mother and fetal erythropoietic tissues is calculated as described by Salvadori et al. (1992), the values obtained are 40.08 and 34.69%, respectively. Salvadori and associates also observed similar percentages of reduction in CP-induced chromosome aberrations in adult mouse bone marrow cells after a 5-day bC pretreatment. MN counts in epithelial cells in sputum of heavy smokers were also approximately 30% lower in the 14-week ßC-supplemented group (Poppel et al., 1992). A relationship between bC doses or plasma level and the reduction of genotoxicity was not observed in these studies. We believe that the protection of CaJ against CP-induced genetic damage observed in this study was mainly due to its carotenoid content. The reduction of genotoxic activity induced by CP observed in vivo and the absence of protection from damage induced by the direct acting agent MMC in vitro led Salvadori et al. (1993) to suggest that ßC affects cytochrome P450 enzyme processing. CP is first oxidized by the microsomal cytochrome P450-linked enzyme to be further converted into its biologically reactive ultimate metabolites, acrolein, phosphoramide mustard and nornitrogen mustard. Phosphoramide mustard alkylates DNA (Mohn and Ellenberger, 1976). In mice the increasing anticlastogenic activity of ßC at lower doses and the absence of a protective effect at higher concentrations suggest different mechanisms of ßC modulation and a possible alteration of the balance of CP activation/detoxification mechanism (Salvadori et al., 1992). ßC reduced the damage induced by CP in cultured human hepatoma cells independent of the treatment regime (pre, post or simultaneous). At higher doses of ßC this effect reached a plateau showing a saturation of the protective mechanisms. At lower doses, a pre- plus simultaneous regime appeared to be more effective, suggesting that ßC can act by inhibiting or inducing activation or deactivation of the promutagen (Salvadori et al., 1993).

The reduction in the frequency of gene mutation induced by the direct acting mutagen ENU in rat spleen lymphocytes was dose dependent but nonlinear within the range of ßC oral doses used. The post-treatment was more effective than were the pre- or the combination of pre- and post-treatments. As ENU can undergo decomposition to yield carbenium ions, this result suggests a scavenging property of ßC or that ßC can affect DNA repair processes (Aidoo et al., 1995). In vivo assays show that ßC protects against the radical-mediated oxidative damage as well as against photoactivated metabolites or xenobiotics and is an effective quencher of single oxygen and other radical species (Rousseau et al., 1992). The hypothesis that ßC protects cells against CP-induced oxidative damage cannot be ruled out, and is supported by the fact that stobadine, a pyridoindole structure with antioxidant properties, protects against CP-induced MN in mother bone marrow and fetal liver (Chorvatovicová and Ujházy, 1995).

Genotoxicity protection seems easy and inexpensive. In mice 0.5 mg/kg of ßC oral gavage for 7 days reduces the frequency of bone marrow mPCE induced by radiation (Abraham et al., 1993). The authors propose that this dose is equivalent to 30 mg/day for a person weighing 60 kg and can be obtained by the intake of 120 g of vegetables rich in ßC. Although the relevance of animal studies to humans remains uncertain our data also suggest that the inexpensive natural anticlastogens present in carrot can contribute to human health.

ACKNOWLEDGMENTS

We thank Dr. Nance B. Nardi for critical suggestions regarding this manuscript. This work was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS) and Financiadora de Estudos e Projetos (FINEP).

RESUMO

O dano genético durante o período pré-natal pode ser uma importante fonte de alterações neoplásicas e de outras doenças durante a vida pós-natal. O beta-caroteno (ßC) é considerado um dos mais importantes anticarcinógenos da dieta e pode proteger células de mamíferos contra eventos genotóxicos. Como a cenoura é a fonte clássica do ßC, nós decidimos testar a modulação do suco de cenoura fresco (CaJ) sobre a genotoxicidade induzida pela ciclofosfamida (CP) no tecido eritropoiético materno e fetal. O tratamento de fêmeas BALB/c com o CaJ ocorreu desde o 7º dia gestacional. Nós observamos, no 16º dia gestacional, que tal tratamento não modificou a freqüência espontânea de eritrócitos policromados micronucleados (mPCE) na medula óssea materna ou no fígado fetal. Quando fêmeas prenhes pré-tratadas com o CaJ receberam uma injeção intraperitoneal de CP (40 mg/kg) no 15º dia gestacional, 24 h depois foi observada uma freqüência de mPCE significativamente menor do que a observada no grupo tratado somente com a CP, tanto na medula óssea materna como no fígado fetal. Estes resultados indicam a presença de anticlastógenos naturais na cenoura.

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(Received January 14, 1998)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  14 Jan 1998