Brief Communications

Inhibitory Effects of Chlorophyllin on MicronucleusFormation Induced by Ethyl Carbamate and Its

Proximate and Ultimate Carcinogenic Forms in MousePeripheral Reticulocytes

Seong Gon Kim,1 Young-Joon Surh,2* and James A. Miller1

1 McArdle Laboratory for Cancer Research, School of Medicine,University of Wisconsin, Madison, Wisconsin

2 Research Institute of Pharmaceutical Sciences, College of Pharmacy,Seoul National University, Seoul, Korea

INTRODUCTION

A variety of plant and vegetable constituents have beenshown to protect against genotoxicity of environmentalcarcinogens and mutagens [Block et al., 1992; Stavric,1994]. Chlorophyll, the ubiquitous pigment present in manygreen plants, has been found to possess strong antigenotoxicactivities [Lai et al., 1980]. Since chlorophyll is insoluble inaqueous solution, the water-soluble sodium-copper deriva-tive chlorophyllin (CHL) has been used experimentally asan alternative to lipophilic chlorophyll. Like chlorophyll,CHL has antimutagenic or anticlastogenic effects compara-ble to those of chlorophyll. Thus, CHL has been shown toinactivate a wide array of mutagens in vivo [Negishi et al.,1994; Guo and Dashwood, 1994; Dashwood et al., 1991;Sarkar et al., 1993; Olvera et al., 1993; Abraham et al.,1994; Renner, 1990] and in vitro [Whong et al., 1988; Onget al., 1986; Camiano et al., 1994; Espinosa-Aguirre et al.,1993]. There have also been limited studies determining itseffects on experimental carcinogenesis [Nelson, 1992;Bronzetti et al., 1990; Park and Surh, 1996]. Arimoto et al.[1993] reported that the antigenotoxic effects of CHL weremost prominent for those mutagens which have a planararomatic ring structure. Because it bears a pyrrole ringsystem, CHL is considered to inactivate aromatic mutagensthrough molecular trapping of their planar structuresthrough strongp–p interactions [Arimoto et al., 1993;Tachino et al., 1994; Breinholt et al., 1995; Dashwood andGuo, 1993]. However, a recent in vitro investigation hasshown that CHL can exert antimutagenicity against ali-phatic carcinogens as well, such as vinyl carbamate (VC)and its ultimate electrophilic form, vinyl carbamate oxide(VCO), probably by complex formation throughp-cationinteraction [Park et al., 1995]. However, it remained largely

unknown whether such detoxification could occur by sys-temic administration of CHL to experimental animals.

As part of our research program to evaluate chemopre-ventive or chemoprotective potential of CHL and its under-lying mechanisms, we determined its effects in vivo onmicronucleus formation induced in mouse peripheral bloodreticulocytes by the typical small nonplanar carcinogenethyl carbamate (EC) and its proximate and ultimate carci-nogenic forms. For comparison, the anticlastogenic activityof CHL was also examined with benzo[a]pyrene (B[a]P) asa reference aromatic mutagen.

MATERIALS AND METHODS

EC and B[a]P were supplied by Sigma Chemical Co. (St. Louis, MO).VC and VCO were synthesized as described previously [Park et al., 1990].CHL was purchased from Aldrich Chemical Co. (Milwaukee, WI). Tri-octanoin was a product of the Pfaltz and Bauer Inc. (Stamford, CT). MaleC3H/HeJ and female C57BL/6J mice from the Jackson Laboratory (BarHarbor, ME) were bred in our laboratory to give B6C3F1 offspring. MaleB6C3F1 mice (8–10 weeks of age) were gavaged twice with CHL (100mg/kg) 1 hr and immediately before a single intraperitoneal injection of EC(500 mg/kg), VC (25 mg/kg), VCO (10 mg/kg), or B[a]P (200 mg/kg). ECand VC were dissolved in phosphate-buffered saline (pH 7.4), whereasVCO and B[a]P were dissolved in trioctanoin immediately before admin-istration. Peripheral blood was collected from the tail vein just before or 24,48, and 72 hr after the clastogen administration and placed on an acridineorange-coated glass slide. The frequencies of micronucleated reticulocyteswere scored according to the method of Hayashi et al. [1990]. Significant

Contract grant sponsor: the Research Institute of Pharmaceutical Sciences,College of Pharmacy, Seoul National University.

*Correspondence to: Professor Young-Joon Surh, College of Pharmacy,Seoul National University, Shinlim-dong, Kwanak-gu, Seoul 151-742,South Korea. E-mail: surh@plaza.snu.ac.kr

Received 5 February 1999; provisionally accepted 12 June 1999; and infinal form 16 June 1999

Environmental and Molecular Mutagenesis 34:57– 60 (1999)

© 1999 Wiley-Liss, Inc.

differences between experimental groups were determined by the Student’st-test. AP-value of,0.05 was regarded as statistically significant.

RESULTS AND DISCUSSION

The micronucleus assay with mouse peripheral bloodreticulocytes has been widely used to evaluate the clasto-genic effects of many potential carcinogens. The assay canmeasure the production of micronuclei as a function of timeusing a small volume of blood [Hayashi et al., 1990; Kishiet al., 1992]. The frequency of micronucleated reticulocytes

(MNRETs) is regarded as a valid index of in vivo clasto-genicity. The intraperitoneal injections of EC and VC re-sulted in considerable increases in MNRETs in peripheralblood of male B6C3F1 mice (Fig. 1A,B). The maximumfrequencies of MNRETs were observed at 48 hr for bothclastogens. The clastogenic potency of VC was about 40times higher than that of EC, which is consistent with theresult from the bone marrow micronucleus assay [Wild,1991]. The oral administration of CHL decreased the fre-quency of MNRETs induced by EC (500 mg/kg) and VC(25 mg/kg) by 25% and 45%, respectively (Fig. 1A,B) at 48hr. VCO (10 mg/kg), the presumed ultimate carcinogenicform of EC and VC, was a stronger inducer of micronucleithan the parent carcinogens (Fig. 1C). It also showed themaximum frequency of MNRET at 48 hr, which was re-duced about 50% by the CHL treatment (Fig. 1C). Thearomatic carcinogen B[a]P (200 mg/kg) caused a continu-ous increase in the formation of micronuclei-containingreticulocytes up to 72 hr (Fig. 2), probably due to slowerelimination of B[a]P in male B6C3F1 mice than the afore-mentioned nonaromatic and less lipophilic carbamates. TheB[a]P-induced MNRET formation reduced about 50% fol-lowing pretreatment with CHL (Fig. 2). Neither trioctanoinnor CHL (100 mg/kg, twice) alone induced the micronucle-ated reticulocytes in peripheral blood. The numeric datasummarizing the effects of CHL on the micronucleus for-mation in peripheral reticulocytes induced by the aforemen-tioned clastogens are collectively shown in Table I.

Since CHL bears a pyrrole ring system, the compoundhas been considered to act as an interceptor molecule capa-ble of trapping various mutagens. CHL exhibited prominentantigenotoxic effects against those mutagens that possess aplanar aromatic ring structure [Arimoto et al., 1993]. Theformation of reversible or noncovalent complexes with ar-omatic mutagens throughp–p interaction has been consid-ered one of the plausible mechanisms underlying the che-

Fig. 1. Anticlastogenicity of CHL against EC and its proximate (VC) andultimate (VCO) mutagenic forms. CHL (100 mg/kg) was administrated p.o.twice to male B6C3F1 mice 1 hr and immediately before the i.p. injectionof EC (500 mg/kg) (A), VC (25 mg/kg) (B), or VCO (10 mg/kg) (C).Peripheral blood was collected at the indicated time intervals after theinjection of each mutagen and the frequency of MNRETs were scored onthe acridine orange-coated slides.

Fig. 2. Anticlastogenicity of CHL against B[a]P. CHL (100 mg/kg) wasadministrated p.o. twice to male B6C3F1 mice 1 hr and immediately beforethe i.p. injection of B[a]P (200 mg/kg). Peripheral blood was collected atthe indicated time intervals after the B[a]P injection and the frequency ofMNRETs were scored on the acridine orange-coated slides.

58 Kim et al.

moprotective effects of CHL. It has been reported to form astrong noncovalent complex with aflatoxin B1 (AFB1) invitro [Breinholt et al., 1995], which might contribute to theinhibition of DNA adduct formation and mutagenesis in-duced by this carcinogen [Dashwood et al., 1991; Breinholtet al., 1995]. The mutagenicity of polycyclic mutagens suchas B[a]P was also mitigated by CHL [Arimoto et al., 1993;Tachino et al., 1994; Dashwood and Guo, 1993]. However,it has recently been suggested that CHL forms a molecularcomplex not only with aromatic planar compounds but alsowith a nonplanar compound such as VC, which appears toaccount for its antimutagenic and antitumorigenic propertiesagainst this carcinogen [Park et al., 1995]. Formation ofDNA adducts by the ultimate electrophilic and carcinogenicform of VC was also markedly attenuated in the presence ofCHL. Likewise, mutagenicity and carcinogenicity of VCOwere inhibited by CHL. Since VCO has no olefinic bond inits structure, it is conceivable that the formation of inactivecomplexes with VCO is mediated by mechanisms other thanp–p interactions. Therefore, the molecular trapping of non-planar compounds (EC and VC),p–p interactions with thearomatic carcinogen (B[a]P) or the nucleophilic addition tothe corresponding ultimate electrophilic and carcinogenicepoxides could (VCO or B[a]P diol epoxide) might contrib-ute to protective effects CHL exerted against the carcino-gens tested in the present study. There are other possiblemechanisms underlying the chemoprotective action ofCHL. Interference with conversion of procarcinogens toultimate carcinogenic metabolites will reduce the chance todamage the target cell DNA and subsequent mutations inthe hot spots. The intraperitoneal administration of CHLand other sodium metallochlorophyllin derivatives were re-ported to lower the activities of several microsomal en-zymes via downregulation of liver hemoprotein followed bythe reduction of the content of microsomal cytochromes[Imai et al., 1986]. In another study, the incubation of CHLwith rat hepatic microsomes in vitro inhibited cytochromeP450IA and P450 IIE1 [Tachino et al., 1994], isoformsresponsible for the metabolic activation of B[a]P and car-bamates (EC and VC), respectively. Nonspecific inhibition

by CHL of cytochrome P450 has also been demonstrated[Yun et al., 1995]. In addition, CHL has been suggested tomodulate the DNA repair process. Thus, posttreatment ofCHL attenuated the clastogenicity of doxorubicin in cul-tured human lymphocytes [Amara-Mokrane et al., 1996].Furthermore, the anticlastogenic effects of CHL has alsobeen attributable to its capability of scavenging free radicals[Sato et al., 1984]. Administration of CHL decreasedg-ray-induced sister chromatid exchange [Morales-Ramirez andGarcia-Rodriguez, 1994; Hayatsu et al., 1993], which maybe associated with suppression of oxidative damage byCHL. One or several of the aforementioned mechanisms cancontribute to the protective effects of CHL against micro-nucleus formation induced by treatment with EC, VC, VCO,or B[a]P in mouse peripheral reticulocytes. Additional stud-ies utilizing diverse types of mutagens and carcinogensshould follow to precisely assess the molecular basis ofchemoprotective or chemopreventive activities of CHL.

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TABLE I. Numerical Summary of Data on Inhibition by CHL of Micronucleus Formation Induced by EC, VC, VCO, andB[a]P in Mouse Peripheral Reticulocytes

Clastogen CHL

MNRETs/1000 RETs measured at

0 h 24 h 48 h 72 h

EC 2 3 6 0.6 36 1.5 246 3.8 106 4.5EC 1 2 6 1.7 26 0.6 186 1.5* 9 6 2.1VC 2 2 6 0.6 46 3.1 266 1.0 86 1.0VC 1 3 6 0.6 56 0.6 186 1.0* 7 6 2.3VCO 2 2 6 0.6 86 2.1 216 4.9 66 0.6VCO 1 2 6 1.2 56 3.5 116 8.0** 5 6 2.9B[a]P 2 2 6 1.0 26 1.2 156 1.5 186 4.4B[a]P 1 2 6 1.0 26 1.0 86 1.2* 9 6 1.7*

*The values are expressed as mean6 SD from three mice. Significantly different from the corresponding counterpart treated with the carcinogen alone(* P , 0.01; **P , 0.05).

Inhibitory Effects of Chlorophyllin 59

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Accepted by—S. Lewis

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