Research Section
Effects of β-Carotene, Retinal, Riboflavin, α-Tocopherol and Vitamins C and K1 on Sister-chromatid Exchanges Induced by 3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and Cyclophosphamide in Human Lymphocyte Cultures

https://doi.org/10.1016/S0278-6915(98)00068-4Get rights and content

Abstract

The vitamins and related compounds cited in the title were investigated for their abilities to modulate sister-chromatid exchanges (SCEs) induced by Trp-P-2 or cyclophosphamide (CP) in human peripheral lymphocyte cultures in the presence of an exogenous metabolizing system from rat liver. When inducer and test substances were given simultaneously, β-carotene, retinal and α-tocopherol caused a dose-dependent decrease of SCE frequencies induced by Trp-P-2 and CP. Vitamin K1, however, brought about an identical effect with Trp-P-2 only, while with CP an initial decrease of SCEs was followed by a statistically significant re-increase at higher concentrations. Vitamin C was ineffective against Trp-P-2, but caused an overall increase of SCEs induced by CP. When blood cultures were preincubated with vitamins before the addition of CP or Trp-P-2, basically identical effects were observed with β-carotene, retinal, α-tocopherol, vitamin K1 and vitamin C. Riboflavin decreased SCEs induced by Trp-P-2 in all treatment schedules, although statistically confirmed minima were observed in the dose–response curves, except in post-treatment experiments. On the other hand, riboflavin only reduced SCEs induced by CP when it was preincubated with lymphocytes. When vitamins were applied in a post-treatment schedule after removal of Trp-P-2 or CP, again, basically identical results against both genotoxins were observed with β-carotene, retinal and α-tocopherol with vitamin K1, however, only with respect to Trp-P-2, and with vitamin C only with respect to CP. In the post-treatment schedule, vitamin K1 caused a decrease of SCE frequencies induced by CP, and vitamin C a decrease of SCEs induced by Trp-P-2.

Introduction

When proteinaceous foods such as fish and meat, and especially beef, are heated, a series of heterocyclic aromatic amines are generated, which may be subdivided into two groups: compounds with a 2-aminoimidazole structure and an additional quinoline (IQ), quinoxaline (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or pyridine (PhIP) ring (IQ-type substances) and compounds with a 2-aminopyridine structure such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), Trp-P-2, 2-amino-6-methyl-dipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1) and 2-aminodipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2); non-IQ-type substances; Commoner et al., 1978; Sugimura et al., 1977). The non-IQ-type substances are generated particularly by pyrolysis of amino acids and proteins. The compounds cited above are all carcinogenic in rodents and mostly potent mutagens in the Salmonella/reversion assay, and were therefore called ‘cooked food mutagens’ (Ohgaki et al., 1991; Sugimura et al., 1977). Other genotoxic effects of these compounds, namely gene mutations, chromosome aberrations, sister-chromatid exchanges (SCEs), DNA strand breaks, and oncogene activation, are in general weakly expressed in mammalian cells (Aeschbacher and Turesky, 1991). With respect to SCEs as an endpoint, nearly all cooked food mutagens induced only low numbers, which were mostly not dose dependent (Aeschbacher and Turesky, 1991; Thompson et al., 1987). Relatively strong and dose-dependent effects were, however, seen with PhIP and Trp-P-2 in Chinese hamster ovary cells (Thompson et al., 1983and 1987).

In accordance with epidemiological studies, which have shown a protective effect of fruit and vegetable consumption against many human cancers (Block et al., 1992; Steinmetz and Potter, 1991), we recently demonstrated protective effects of fruits and vegetables (Edenharder et al., 1994), as well as of pure compounds such as vitamins and flavonoids (Edenharder et al., 1993; Edenharder and Worf-Wandelburg, 1991), against mutagenicity induced by IQ and other heterocyclic aromatic amines of this class in the Salmonella/reversion assay. Again, the bulk of data available for protective effects of natural compounds of plant origin and other substances as well refer to mutagenicity in bacterial test systems (Hartman and Shankel, 1990; Hayatsu et al., 1988). Information about inhibition of genotoxicity by these compounds in other in vitro and in vivo test systems such as SCEs, chromosomal aberrations, and the micronucleus test, as well as in carcinogenicity experiments is, however, limited. We therefore decided to investigate whether vitamins found active against several cooked food mutagens, among them Trp-P-2 in Salmonella, would prove protective also against SCEs induced by Trp-P-2 in cultured human lymphocytes. Other (pro)vitamins, although found inactive or only marginally active in the Salmonella/reversion assay such as β-carotene, ascorbate and α-tocopherol, were included because of their general importance for the human and protective effects in other test systems. In addition to Trp-P-2, we also investigated all these (pro)vitamins for protective effects against induction of SCEs by CP. Reasons for this were: CP is also of real relevance to the human since it is a cytostatic drug, frequently used for patients with tumours. Furthermore, typical for most carcinogens, both CP and Trp-P-2 need metabolic activation, but the pathways are quite different. CP consistently induces high numbers of SCEs and was widely used for cytogenetic investigations in the past. Inclusion of CP in our investigations therefore enabled comparisons which would not have otherwise been possible.

Section snippets

Chemicals

Cyclophosphamide, β-carotene, retinal (vitamin A-aldehyde), vitamin K1 (2-methyl-3-phytylnaphthoquinone), and bisbenzimide H 33258 were purchased from Serva Biochemicals (Heidelberg, Germany). Trp-P-2 acetate (3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate) was from Wako Chemicals (Neuss, Germany). dl-α-tocopherol and ascorbic acid were obtained from Merck (Darmstadt, Germany). Chromosome-medium B, Hanks’ salt solution (without Co2+, Mg2+) and colcemide solution were purchased from Nunc Chemie

Results

As can be concluded from Table 1, Trp-P-2 as well as CP in the presence of an exogenous metabolizing system from rat liver (S-9) induced a dose-dependent increase in SCEs in cultured human lymphocytes. In the concentrations used in these investigations—10−5 m for Trp-P-2 and 10−4 m for CP—the two compounds caused about a five- and eightfold increase in SCEs over that of the controls, 3.4±1.4 and 3.15±1.4 SCEs/cell, respectively. Of the vitamins investigated by us for protective effects against

Discussion

When investigated in a pre-, simultaneous, or post-treatment schedule for protective effects against SCEs, induced by Trp-P-2 or CP in cultured human lymphocytes, six different members of the heterogeneous group of organic compounds known as vitamins produced a complex response pattern, as can be derived from the data presented above. Relatively simple situations were observed with β-carotene, retinal and α-tocopherol: In most cases, β-carotene, retinal and α-tocopherol caused highly

References (41)

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