Detection of mutagenic polycyclic aromatic hydrocarbons in african smoked fish

SHORT DETECTION AROMATIC

PAPERS

OF MUTAGENIC HYDROCARBONS SMOKED FISH

POLYCYCLIC IN AFRICAN

K. MOSSANDA, F. PONCELET*, A. FOUAWN and M. MERCIER* Lahoruroire *Lohorctror.v

(f A~u/.Iw t/es Denrk Alirvenrairrs. Unicersiti Boulerard de lu Constirurion, 151. 8.4010 LiGgr o/ fJioro.ricology. Unirersir!, 01 Loutwin. School UCL-73.69, B. I-700 Brussels. Belgium (Receiced

2 October

dr LiPge.

and of Pharmacy.

19711)

Abstract-The mutagenicity of six polycyclic aromatic hydrocarbons found in African smoked fish was tested using several Salmonella ryphimurium strains. In the presence of fortified rat-liver post-mitochondrial fractions, mutagenic activity was observed with o-phenylenepyrene, coronene, benzo[g,h,i]perylene and triphenylene in the plate incorporation method, and with fluoranthene in the bacterial fluctuation test. No mutagenic effects of benzo[b]fluoranthene towards any of the tested strains were detected.

Cg,h,ilperylene (C), o-phenylenepyrene, anthanthrene, coronene, dibenz[a,c]anthracene (C,M), benzo[b]tetraphene (C,M), dibenz[a,h]anthracene (C,M), dibenzo[a,l]pyrene (C,M). Since the mutagenic activity of some of these compounds was still unknown, six compounds were tested using the S. typhimurium strains. The compounds tested were: coronene, benzo[g,h,i]perylene, triphenylene, fluoranthene, o-phenylenepyrene and benzo[b]fluoranthene.

Introduction

The high incidence of stomach cancer among populations living in Iceland and in Baltic countries has been causally related to the intensive consumption of smoked fishes by people from those countries (Bailey & Dunjal, 1958). Various polycyclic aromatic hydrocarbons (PAHs) have been isolated from industrially smoked fish (Bailey & Dunjal, 1958; Grimmer & Hildebrandt, 1967; Howard, Teague, White & Fry, 1966; Lijinsky & Shubik, 1965). However. woodsmoked fish were less extensively investigated although comparatively high levels of benzo[a]pyrene (37pg/kg) have been detected in such fish (Masuda & Kuratsune, 1971). Many PAHs that are carcinogenic in rats have been isolated from cigarette smoke condensates (Akin, Snook, Severson. Chamberlain & Walters. 1976); moreover. carcinogenicity and mutagenicity tests have been carried out on the various fractions obtained after chromatography of such condensates on a silica gel column (Bock, Swain & Stedman, 1970; Kier, Yamasaki 8~ Ames, 1974). Recently, the skin from smoked fishes, smoked meats and the smoke condensate used for their processing (Nagao. Honda, Seino, Yahagi & Sugimura, 1977) were shown to be mutagenic towards Sabt~onrlla ryphimurium. For a large proportion of people from Central Africa, an essential part of their dietary protein is obtained from fish smoked by an artisanal wood combustion procedure. Twenty PAHs have been isolated and identified in smoked fish from Zaire (K. Mossanda, unpublished data, 1978). Some of these are reported to exert various carcinogenic (C) effects in animals (Dipple, 1976; Hoffmann & Wynder. 1976) and, or mutagenic, (M) effects towards S. ryphimurium strains (McCann, Choi, Yamasaki & Ames, 1975) as indicated below: anthracene (M), phenanthrene (M), Ruoranthene, pyrene (M), chrysene (C,M), benz[trlanthracene (C,M), triphenylene. benzo[a]pyrene (C,M), benzo[h]fluoranthene (C), benzob]fluoranthene (C), perylene, benzo [elpyrene (CM), benzo-

Experimental Materials. Coronene and benzo[g,h.i]perylene were obtained from Fluka (Leuven); triphenylene, fluoranthene and o-phenylenepyrene from Aldrich Europe (Beerse), and benzo[b]fluoranthene from Koch-Light Laboratories Ltd. (Brussels). All other products were of the purest grade commercially available. PAHs were stored in the dark at 4°C in the presence of gaseous nitrogen. Dilutions were made in dimethylsulphoxide (DMSO). Animals. Adult male Wistar rats (20&25Og) were fed an RAL diet. The animals were injected ip (dosage; 5OOmg/kg) with Arochlor 1254 diluted in corn oil (200 mg/ml) 5 days before the preparation of the liver fractions (Ames, McCann & Yamasaki. 1975). Mutagenicity assays. S. typhimurium strains TAl530, TAl535, TA1537, TAl538, TA98 and TAlC0 were kindly provided by Professor B. N. Ames. The post-mitochondrial (S9) fractions were obtaihed from three pooled rat livers, the homogenate (3 ml of 015 M-KCl/g wet liver) of which was centrifuged by the classical procedure (Ames er al. 1975). The S9 mix was prepared according to Ames et al. (1975) by adding MgClz (8pmol/ml mix), KCI (33pmol/ml mix), sodium phosphate (I 00 ~mol/ml mix),. glucose-6phosphate (5 pmol/ml mix) and NADP+ (4 pmol/ml mix). Either 100~1 (25 mg wet liver)/ml or 300~1 (75 mg wet liver)/ml mix of S9 were used. Plate tests were performed in duplicate by mixing substrate dilutions (0.1 ml/plate), 2-8 x 10’ bacteria

I41

K. MOSSANIIA. (b) 4Oi.

F. PONCELICT. A. FOUASSIN

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.

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4 .a

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Q a

8

-

Concanfrotion.

g

~g/plata

807

. I x

x

x

,

50

ConcenhWion.

jq&laE?

Cd)

0 0 0

0

0 >

a

0

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20

Fig.

1. Mutagenic activity of PAHs towards S. ~yphimurium strains TA1538 (0). TAlOO (o), TA1537 (o), TA98 (x) in the presence of S9 mix Arochlor 1254 (100~1 S9/ml mix);

(a) o-phenylenepyrene, benzoCg.h,i]perylene.

(b) triphenylene.

cc) coronene, (d)

from an overnight culture in nutrient broth (Difco)/ plate. and S9 mix (05 ml/plate) in histidine-biotin (0.05 mM)-supplemented top agar (2 ml/plate), which was layered on minimal glucose agar (Vogel Bonner E medium) in Petri dishes. The plates were incubated for 48 hr at 37°C in the dark and the numbers of his’ revertant colonies were calculated. The toxicity

Substrate Polycyclic

dilution

aromatic

Results

No mutagenic effect was detected with benzo[b] fluoranthene towards any of the tested strains. In the presence of S9 mix, mutagenic activity was observed with o-phenylenepyrene, coronene. benzo[g.h.i]perylene and triphenylene by the plate incorporation method (Fig. 1). Under these conditions. no cytotoxic effects of PAHs were detected. With o-phenylenepyrene, the strains TAlOO, TA1537 and TA1538 reverted at concentrations in the region of 3 pg/plate. TA 100 his+ revertants increased considerably with triphenylene at lOpg/plate or more. With coronene,

Average no. of positive tubes per rack

Strain

Benzo[b]fluoranthene

TA98

10 loo

21

TAIOO

10 loo

23.6

TA98

50

18.3

Triphenylene

ControI

100 TAIOO

Benzo[g.h,i]perylene

TA98 TAIOO TA1538

Coronene

TA98 TAIOO

o-Phenylenepyrene

TA98

Fluoranthene

TA98 TAIOO

NS = Not significant

50

100 I IO I 10 I IO

M. MERCIEH

of the substrate was evaluated by determining the bacterial survival with a lower bacterial inoculum (104-fold dilution) and plates of nutrient agar (Difco). Bacterial fluctuation tests were performed in triplicate using a modification of the method proposed by Green. Bridges, Rogers, Horspool. Muriel, Bridges & Fry (1977). Into a sterile tube kept in iced water, were successively introduced: liquid minimal glucose medium (Vogel-Bonner E medium) supplemented with histidine and biotin (0.005 mM) (4 ml), 2-8 x 10’ bacteria from an overnight culture in nutrient broth, substrate dilution in DMSO (0.1 ml), and S9 mix the composition of which was 300~1 S9/ml mix (1 ml). After homogenization, the mixture was distributed at a constant volume of 0.1 ml/tube in 50 sterile tubes. The tubes were then incubated at 37°C in the dark for 3 hr. Histidine-biotin-supplemented liquid minimal glucose medium (2 ml) containing bromocresol purple (BCP) (5pg/ml) was added to each tube. The incubation was then carried on at 37°C until a total incubation time of 72 hr had elapsed. The numbers of growing positive tubes/rack were counted.

hydrocarbon

(s/ml)

and

36.3 17.3 33 19.6

2 20 2 20 0.5 5

26.6

IO loo IO 100

18.3

18.3 23

293.

Treated 20.3 18 24.6 30.6 26.6 30.3 36 41.3 21.3 27 33.6 39.3 15 31.3 32.6 38 18.3 19.3 31.6 32 22.6 46.3 26.3 50

Significance (P)

NS NS NS NS NS <0,05

NS NS NS <0,05

NS NS NS
NS io.05

NS NS NS NS NS -co.01 NS
Mutagenicity

of PAH from smoked fish

TA1538 and TA98 were the most sensitive strains: a reversion to histidine prototrophy was noticeable at a concentration of 1 pg/plate. With benzo[g,h,i]perylene, a concentration of Zpg/plate caused the strain TA1537 to revert. By utilizing the bacterial fluctuation test (Table I), weakly positive effects were observed with triphenylene (TA98). benzo[g,h,i]perylene (TA98. TA 1538) and coronene (TA98). With fluoranthene. a highly significant positive response (P < 0.01) was obtained with the strains TA98 and TAlOO although this latter compound showed no mutagenic activity in the classical plate incorporation method. Discussion Because of their poor water solubility. these PAHs were generally tested at a narrow range of concentrations. Nevertheless. the doses where an increasing reversion to prototrophy was observed (l-10 pg,‘plate) were of the same order as the doses of other common PAHs such as benzo[a]pyrene that are active. Moreover, the strains sensitive towards these compounds were those on which many PAHs have already demonstrated a positive mutagenic effect (McCann er ol. 1975). These results point out the possible longterm deleterious effects associated with the presence of PAHs in the human environment and particularly in some smoked foods. REFERENCES

Akin. F. J., Snook. M. E., Severson. R. E., Chamberlain, W. J. & Walters. D. B. (1976). Identification of polynuclear aromatic hydrocarbons in cigarette smoke and-their importance as tumorigens. J. narn. Cancer Insr. 57. 191. Ames. B. N.. McCann. J. & Yamasaki. E. (1975). Method for detecting carcinogens and mutagens with the Salmicrosome mutagenicity test. monella/mammalian Maration

Rrs.

31. 347.

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Bailey, E. J. & Dunjal. N. (1958). Polycyclic hydrocarbons in Icelandic smoked food. Br. J. Comer 12. 348. Bock, F. G.. Swain, A. P. & Stedman. R. L. (1970). Composition studies on tobacco. XLI. Carcinogenesis assay of subfractions of neu’tral fraction of cigarette smoke condensate. J. narn. Cancer fnsr. 44. 130.5. Dipple, A. (1976). Polynuclear aromatic carcinogens. In Chemical Carcinogens. Edited by C. E. Searle. p. 245. ACS monograph no. 173. Washington D.C. Green M. H. L., Bridges, B. A., Rogers. A. M., Horspool, G., Muriel, W. J. Bridges, J. W. & .Fry, J. R. (1977). Mutagen screening by a simplified bacterial fluctuation test: use of microsomal preparations and whole liver cells for metabolic activation. Muration Res. 48, 287. Grimmer, G. & Hildebrandt, A. (1967). Kohlenwasserstofie in der Umgebung der Menschen. V. Der gehalt polycyclisher koklenwasserstoffe in fleisch und Raiicherwaren. 2.

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69, 223.

Hoffman, D. & Wynder, E. L. (1.976). Environmental respiratory carcinogenesis. In Chemical Carcinogens. Edited by C. E. Searle. p. 325. ACS monograph no. 173, Washington D.C. Howard, J. W.. Teague. R. T.. White. R. H. & Fry. B. E. (1966). Extraction and estimation of polycyclic aromatic hydrocarbons in smoked foods. 1. General method. J. A~I. Oil Chem. Sot. 49, 596. Kier. L. D.. Yamasaki. E. & Ames, B. N. (1974). Detection of mutagenic activity in cigarette smoke condensates. (Carcinogenesis/Salmonella tester strains/microsomal activation). Proc. narn. Acad. Sci. 71, 4159. Lijinsky. W. & Shubik. P. (1965). Polycyclic hydrocarbons carcinogens in cooked meat and smoked food. Ind. Med. Surg. 34. 152. McCann, J., Choi, E.. Yamasaki, E. & Ames, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals. Proc. narn. Acud. Sci. 72. 5135. Masuda. Y. & Kuratsune. M. (1971). Polycyclic aromatic hydrocarbons in smoked fish “katsuobushi”. Gann 62. 27. Nagao, M.. Honda. M.. Seino. Y.. Yahagi. T. & Sugimura. T. (1977). Mutagenicities of smoke condensates and the charred surface of fish and meat. Cancer Lerr. 2. 221.