Food and Chemical Toxicology 35 (1997) 567-571 1~ mtck,,,~!
Sister Chromatid Exchanges Induced In Vitro and In Vivo by an Extract of Black Pepper E. M A D R I G A L - B U J A I D A R * t , S. D I A Z B A R R I G A : ~ , P. M O T A t , R. G U Z M A N ~ a n d M . C A S S A N I t tLaboratorio de Gen6tica, Escuela Nacional de Ciencias Biol6gicas, I.P.N. Carpio y Plan de Ayala C.P., 11340 M~xico, D.F., M6xico and SLaboratorio de CitogenOtica, Facultad de Estudios Superiores Cuautitl~in-UNAM, M6xico (Accepted 6 December 1996) Abstract--Black pepper is a spice widely used in human food. The aim of this investigation was to determine whether an alcoholic extract of the mature berries of black pepper induced genotoxic damage in vivo and in vitro. The first aspect was evaluated in mouse bone marrow cells and the second one in human lyr~phocytes. In both cases the rate of sister chromatid exchange (SCE) and the replicative index were deterrained. For the in vivo assay, ip doses of 7.0, 14.0, 28.0 and 56.0 mg/kg body weight were tested, with the following results: (1) a significant increase of SCE frequency in all doses tested compared with the control level (the highest dose produced almost a duplication of the basal rate of SCEs); (2) a similar pattern with regard to cell proliferation kinetics at all doses tested, without significant differences between them. For the in vitro assay, doses of 25.0, 50.0, 75.0 and 100.0 #g/ml were tested, with the following results: (1) a significant increase in the frequency of SCEs at all doses tested; a linear regression analysis of the data produced a correlation coefficient of 0.98; (2) a significant reduction in the replicative index, at the two high doses. These results demonstrated that the extract of black pepper was genotoxic in both systems. ~, 1997 Elsevier Science Ltd
A bbreviati~,nAGT s: = average generation time; BrdU = 5-bromodeoxyuridine; CPK = cell proliferation kinetics; Ll)s0 = median lethal dose; M~ = first cellular division; M2 = second cellular division; third M3 =
cellular division; P1 = proliferation index; SCEs = sister chromatid exchange.
INTRODUCTION Piper nigrum, commonly known as black pepper, is a plant of the family Piperaceae, the dried and mature fruits of which are widely consumed as a condiment in human food. The berries are reticulated, almost spherical structures possessing characteristic aromatic and pungent qualities (Evans and Trease, 1991; Maistre, 1967). P. nigrum is used as a preservative in the processing of industrialized meats, and its oil has been used in the manufacture of ice cream, candies and baked goods (Evans and Trease, 1991; Maistre, 1967). In studies of black pepper, Farkas et al. (1981) detected no mutagenic response in the in vitro Salmonella/microsome test using acetone or ether extracts from a spice mixture containing 14% pepper, nor in the in vivo assay of urine metabolites from rats fed for 6 days with the same mixture or with black pepper extracts alone, using five strains of Salmonella-histid:ine auxotropic organisms (TA1535, TA1537, TA1538, TA98 and TA100). Osawa et al. (1981) found a mutagenic response in the Salmonella
*Author for correspondence.
typhimurium assay system, but only when they tested the permitted level of nitrite with the usually accepted amount of pepper; their system included the reaction of sodium nitrite (120 ppm) with the spice in an aqueous solution at pH 3.5 for 20 hr; subsequently, the mutagenicity of ethyl acetate extracts was tested by the Ames assay using strain TA100 without metabolic activation. This result was recently confirmed by Higashimoto et al. (1993), who found no mutagenic effect in the same test system, when aqueous, methanolic and hexane extracts of P. nigrum were tested alone, but who observed a positive response when the first two above-mentioned extracts were treated with nitrite (strain TA100, without metabolic activation). These two reports suggest the possibility of mutagen formation in the human stomach, related to P. nigrum consumption. The clastogenic potential of black pepper has also been investigated with positive results (Abraham and John, 1989). R o o t tip cells of the bean Viciafaba were treated with distilled water extracts of black pepper to determine its effect on cytotoxicity and chromosomal aberrations. The results showed a decrease of mitotic index with high doses (from 100/~g/ml) and an increase in the frequency of
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E. Madrigal-Bujaidar et al.
structural aberrations (chromatid and chromosome types), even with doses as low as 2 #g/ml. In mouse bone marrow cells studied in vivo (John and Abraham, 1991), the authors detected elevations in the number of chromosome breaks and polyploidy in animals treated for 30 days with an aqueous extract. This report is congruent with a study measuring micronucleus formation in mice treated for 6 wk with an ethanolic extract of the spice, which showed a significant genotoxic response starting from wk 3 (Colunga et al., 1994). The reports mentioned above suggest the desirability of extending studies about black pepper genotoxicity; therefore, the aim of this investigation was to evaluate the effect of a black pepper extract on the production of sister chromatid exchanges (SCEs) in human lymphocytes and in mouse bone marrow cells.
MATERIAL A N D M E T H O D S
Preparation o f black pepper extract
Mature berries were obtained from the main distribution centre of Mexico City and were macerated in ethanol (1 g/2 ml). They were then ground in a micromill with stainless-steel cup and blade, and the disintegrated fruits were kept in the dark, at room temperature, for 48 hr. The mixture obtained was vacuum filtered and freezedried. Finally, the extract was dissolved in a 50°,/o solution of dimethyl sulfoxide and distilled water with the help of a sonicator. The procedure described was initially used by Concon et al. (1979) in their studies of the carcinogenic potential of black pepper. In vitro stud),
The blood of a 24-yr-old, non-smoker female donor was used for the experiment. Duplicate lymphocyte cultures were established with 0.6 ml whole blood, 8.5 ml McCoy 5A culture medium (In Vitro), and 0.5 ml phytohaemagglutinin. After 24 hr at 37°C, 5-bromodeoxyuridine (BrdU; Sigma, 15/~g/ml) was added to the cell cultures, as well as 0.0, 25.0, 50.0, 75.0 and 100.0 pg extract/ml. These doses were selected after preliminary assays to determine cytotoxicity produced by the extract. Colchicine (Sigma, 0.5/~g/ml) was added at 71 hr of cell culture and 1 hr later a hypotonic shock with 0.075 M KC1 was applied for 30 min; the fixative (methanol acetic acid, 3:1, v/v) was subsequently added three times. The sister chromatid staining procedure was as previously described (Madrigal-Bujaidar et al., 1991), according to the Fluorescent-plus Giemsa method initially reported by Perry and Wolff (1974). Microscopic scoring to determine the frequency of SCE was done in 30 second-division metaphases per dose, and the statistical significance of this parameter
was initially assessed with an ANOVA test, followed by a Tukey test. The cell proliferation kinetics (CPK) were established in 100 cells per dose, scoring the rate of first (Mr), second (M2) and third (Ms) cellular division. With the data obtained, the proliferation index (PI) was determined using the formula 1M~ + 2M2 + 3M3/100 (Madrigal-Bujaidar et al., 1991). This parameter evaluated statistically with the chi-square test. In vivo stud)'
For the genotoxic study we used male mice (NIH) from the National Institute of Hygiene with a mean weight of 25 g. The animals were kept in polypropylene cages at 23°C and allowed freely to consume standard food (Purina) and drink tap water during the experimental process. An ip LDs0 value was determined following the method described by Lorke (1983); the obtained value (ll3.13mg/kg body weight) served as an indicator to establish the experimental doses used. A 50 mg BrdU tablet partially coated with paraffin was implanted sc in each animal (five mice per lot), and an ip injection of the test substance or its vehicle was given 1 hr later at dose levels of 0.0, 7.0, 14.0, 28.0 and 56.0 mg/kg body weight. 21 hr after the implantation of the tablet, we injected colchicine (Sigma, 8 mg/kg), and 3 hr later the animals were killed by cervical dislocation. The bone marrow cells from both femurs were obtained and kept in 0.075 M KC1 at 3TC for 30 min; finally, the fixative (methanol acetic acid, 3:1, v/v) was added and changed twice. Slides were made by flaming, and chromatid differential staining was performed with the technique mentioned for the in vitro study. Microscopic observations included 25 second-division mitoses per mouse to determine the frequency of SCE. The data obtained were statistically evaluated with an ANOVA test followed by a Tukey test. In addition, 100 cells per animal were scored to establish the CPK and the average generation time (AGT) using the formula 21/(IM~ + 2M2 + 3M3) 100 (Ivett and Tice, 1984). This measurement was statistically evaluated with the chi-square test. RESULTS From the black pepper alcoholic extraction, we obtained 10#g dry matter/ml ethanol, which approximately corresponded to 20.8 mg dry matter extracted per gram of pepper. Data obtained in the in vitro study showed a dose-related SCE increase over the whole range of doses tested. The elevation in the four tested doses was statistically significant with respect to the control level (P = 0.01), and reached a difference of 5.9 SCEs between the high dose (100 ~g/ml) and the control value (Table 1). A linear regression analysis of the data showed a correlation coefficient r = 0.98 (Fig. 1). Table 1 also shows a cell cycle delay
SCEs induced by black pepper extract
Table 1. Frequencyof sister chromatid exchange (SCE) and cell proliferationkinetics(CPK) in human lymphocyteculturestreated with an extract of black pepper D,ase (,ug/ml)
Control 25 50 75 100
7.5 + 0.42 9.94 __+0.58* 10.36 + 0.34* 12.2 _+ 0.57* 13.4 _+ 0.57*
27 33 33 57 69
36 40 43 32 23
37 27 34 11 8
PI? 2.1 1.94 2.2 1.54"* 1.39"*
SCE data were obtained by scoring 30 cells per dose (values are means + SEM), and CPK data were obtained by scoring 100 cells per dose. ?Proliferation index (PI) = (1M~ + 2M: + 3M3)/100, where M~, M2 and M3 represent the frequency of cells in first, second, and third cellular division, respectively. Asteri~;ks indicate significant differences from control (*P = 0.01; **P = 0.01; ANOVA and Tukey tests; chL-square test).
Table 2. Frequency of SCEs and cell proliferation kinetics (CPK) in mice administered with an extract of black pepper Dose (mg/klg)
0.0 7 14 28 56
2 6 2-9 3-10 4-9 5-10
SCE/cell 3.58 5.5 5.5 6.0 6.63
+ 0.07 + 0.05* _+ 0.08* + 0.13" + 0.11"
A G T t (hr)
28 25 27 27 25
51 53 58 54 63
21 22 15 19 12
11.27 11.00 11.13 11.29 11.13
SCE data were obtained by scoring 25 cells per animal (five mice per dose; values are means + SEM). C P K data were obtained by scoring 100 cells per animal (five mice per dose). ?Average ge:aeration time (AGT) = [21/(1M~ + 2M2 + 3M3)]x100, where M~, M2 and M3 represent the frequency of cells in first, ~;econd, and third cellular division, respectively. Asterisks indicate significant differences from control (*P = 0.01; ANOVA and Tukey tests).
induced by the two high doses of the extract--a modification mainly represented by the accumulation of first-division c,ells. According to the LDs0 obtained (113.13 mg/kg body weight), in our in vivo genotoxic study the lowest and highest tested doses corresponded to one-sixteenth and one-half of the LDs0, respectively. As with the in vitro research data, a significant increase in SCE was observed with all doses tested, although withoul: a constant dose-related increase between them and with a maximum net increase of 3.05 SCEs, corre:~ponding to the high dose (56 mg/ kg) in relation to the control level (Table 2). Concerning the CPK, there were no differences between any of the doses tested, as indicated by similar AGT values (Table 2). DISCUSSION
Our results are in agreement with the previously described clastogenic effects induced by black pepper (Abraham and John, 1989; John and Abraham, 1991). However, if we compare our mouse SCE assay with that performed by John and Abraham (1991) and Colunga et al. (1994) to study chromosomal aberrations and micronuclei in the same organism, it seems that SCE i,; more sensitive for the detection of genotoxic injury caused by black pepper. An SCE is an interchange between homologous loci of DNA on sister chromatids, whereas DNA lesions leading to chromosomal aberrations most probably are related to a double-strand break, which may be restituted, may participate in an exchange or may generate a
deletion (Moore and Bender, 1993); our study therefore sheds light on another facet of Piper nigrum genotoxicity. In addition, the use of human lymphocytes in vitro may be an appropriate assay to measure genotoxic potential, considering that it is difficult to evaluate directly the effect of black pepper on consumers, owing to the probable interference from other food components. The analyses of reports on clastogenicity and SCEs, together with studies performed on the bacterial mutagenicity of black pepper extracts, suggest a better expression of its genotoxic potential at the chromosome level than at the gene mutation level. This conclusion seems to be supported by the fact that gene mutations are not expressed p e r se, even when black pepper was mixed with six other spices (Farkas et al., 1981), but they appear only on the addition of an oxidant chemical such as nitrite. The mutagenicity caused by this interaction may be due to the formation of N-nitrosopiperidine, as well as the action of mutagens derived from nitrite-treated piperine--more specifically, the piperic acid moiety of piperine (Osawa et al., 1981). Our positive results are also in accordance with observations about the carcinogenic potential attributed to the condiment. In this field, since the initial report of Concon et al. (1979) on a study in toads, those findings have been confirmed in the same organism as well as in the mouse. These studies have demonstrated that black pepper produces liver tumours (hepatocellular carcinomas, lymphosarcomas and fibrosarcomas), and metastatic deposits in various organs (El Mofty et al., 1988 and
E. Madrigal-Bujaidar et al.
1991; Shwaireb et al., 1990). The above-mentioned results are also supported by studies evaluating risk factors in areas with elevated frequencies of oesophageal and nasopharyngeal cancer, which include the consumption of tannins and black pepper (Ghadirian, 1987 and 1993; Jeannel et al., 1990). Nevertheless, it should be noted that at least two reports in humans did not show important damage to the gastric mucosa, as assessed by the effect of black pepper on the rate of exfoliation of the surface epithelial cells (Desai and Kalro, 1985) or the fractional recovery of gastric or sodium secretions (Myers et al., 1987). The different alkaloids present in black pepper have been identified, including the chemicals piperine, chavicine, piperettine and myristicine (Abraham and John, 1989). Piperine is the major bite factor of the spice, and is also known to possess high toxicity in different mammals (Piyachaturawat et al., 1983). This compound acts as a promoter of the genotoxic damage produced by benzo[a]pyrene, by increasing the binding of its epoxide to DNA (Chu et al., 1994), and also potentiates the hepatotoxicity of carbon tetrachloride by accelerating its biotransformation, thereby increasing lipid peroxidation (Piyachaturawat et al., 1995). Considering these actions, it seems likely that piperine is partly responsible for the results observed. Other components of the spice also have carcinogenic potential by themselves: safrole and tannic acid were considered weak carcinogens when they were compared with the effect of methylcholanthrene (Wrba et al., 1992). Safrole is known to induce intrachromosomal recombination in yeast, unscheduled DNA synthesis in rat hepatocytes, and chromosomal aberrations and 6-thioguanine resistant mutations in V-79 cells, but it fails to induce bacterial mutagenicity and SCEs in mammalian cells in vitro (Carls and Schiestl, 1994; Howes et al., 1990; Jain, 1989; Schiestl et al., 1989). An interaction between the components of the tested mixture is also possible; in this context, Neal (1979) has demonstrated synergism in the toxicity of safrole and myristicine with other chemicals, and a similar mechanism has been described with respect to piperine (Hodgson et al., 1995). It is evident from epidemiological and experimental studies that a number of natural products may cause mutagenicity and cancer in humans; therefore, it seems important to identify potential mutagens/ carcinogens, since it may enable appropriate action to be taken to prevent or reduce the genetic hazards of environmental mutagens. Our study showed a certain genotoxic potential of an extract of black pepper (tested at dose levels higher than those commonly consumed by humans), and it agrees with the increased number of micronuclei detected with the same extract, in mice orally treated with low doses for 6 wk (Colunga et al., 1994). These results suggest the advisability of moderating the consumption of the spice. However, to achieve a balanced conclusion
about its use, its beneficial properties, such as its previously detected antimutagenic/anticarcinogenic capacity (Hashim et al., 1994; Higashimoto et al., 1993), should be borne in mind.
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