J Sci Food Agric 1996,72,455-460

Detection of Immunomodulating Activities in an Extract of Japanese Edible Seaweed, Lawtinaria japon ica ( M akon bu) Yasuji Okai,"* Shigeaki Ishizaka,b Kiyoka Higashi-OkaP and Uki Yamashitac a Department of Human Life Science, Osaka Kun-ei Women's College Showjaku, Set's City, Osaka 566, Japan * Department of Parasitology, Nara Medical University, Shijho-cho, Kashihara, Nara 634, Japan

Minami-Ku, Hiroshima 734, Japan

(Received 18 December 1995; revised version received 18 March 1996; accepted 25 June 1996)

Department of Immunology and Parasitology, Hiroshima University, School of Medicine, Kasumi,

Abstract: A significant immunomodulating activity was found in the hot-water- soluble extract of the most popular edible seaweed in Japan, Laminaria japonica (Makonbu in Japanese) which showed an enhancing activity for DNA synthesis of spleen cells from endotoxin-nonresponder, C3H/HeJ mice. This activity was divided into polysaccharide and non-polysaccharide fractions and the former fraction exhibited much higher activity than that of the latter fraction. The poly- saccharide fraction caused stimulatory effects on the ingestive activity of mouse phagocytic cells against Staphylococcus aureus and the release of cytokines, interleukin-la and tumour necrosis factor a from the same cells. Furthermore, the polysaccharide fraction exhibited enhancing effects on polyclonal antibody (IgM and IgG) production in spleen cells. These immunomodulating activities were associated with polysaccharides themselves, but not contaminating endo- toxins in the fraction judged by comparative experiments. The significance of this finding is discussed from the viewpoint of the immunopotentiation by edible sea- weeds in host animals.

Key words : immunomodulating activities, seaweed, Laminaria japonica

INTRODUCTION incidence of these diseases (Statistical Information Department 1988) eat the highest level of pork meat in Japan but also the largest amount of seaweeds, espe- cially Laminaria japonica (Makonbu) (Statistical Bureau 1991).

Generally, in Japan, various kinds of seaweeds have been used traditionally as an additive or seasoning in cooking various foods. Among the edible seaweeds in Japan, Laminaria japonica (Makonbu) has become the most popular. However, the effect of this seaweed on the immunological regulation in host animals has not hitherto been analysed.

In this paper we describe immunomodulating activ- ities in the hot-water-soluble extract of Laminaria japon- ica in in uitro experiments which demonstrate the enhancing effects of the polysaccharides in the extract

Epidemiological studies showed that the major, but not all, causes of serious diseases such as cancer and heart diseases are associated with environmental factors such as foods or lifestyle (Willet 1994). For example, a com- parative study of some countries showed a positive relationship between the amount of meat or fats and the incidence of some diseases. On the other hand, an inter- esting issue about the relationship between food con- sumption and lifespan or the healthy state of people has been found in comparative data of different regions of Japan. The people in a southwestern seashore district, Okinawa who show the longest lifespan and the lowest

* To whom correspondence should be addressed.

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain 455

456 Y Okai et a1

on some functional activities of the immunocompetent cells of endotoxin-nonresponder mice.

MATERIALS AND METHODS

Preparation of seaweed extract and fractionation of the polysaccharide in the extract

Laminaria japonica was harvested in the Hokkaido dis- trict in Japan and washed extensively with cold deion- ised water and dried. Five grams of dry fronds of L japonica were minced briefly by an electric cutter appar- atus (IFM-100, Iwatani Co, Tokyo, Japan), added to 100 ml of hot distilled water and allowed to cool to room temperature for 30 min. The seaweed extract was recovered by a centrifugation at 1500 x g for 15 min and stored in a freezer at -20°C. The preparation of the polysaccharides from this seaweed extract was carried out according to the previous method (Maeda and Nisizawa 1968). After thawing, 10 ml of the extract was adjusted to 0.3 M NaCl, mixed with three volumes of cold ethanol, kept overnight at -20°C and centri- fuged at 5000 x g for 5 min. The precipitate was dis- solved with 10 ml of deionised water and dialysed using a membrane with a cut-off size of 13 kD (Spectrum Medical Industries Inc, Los Angeles, CA, USA) against 1 litre of deionised water overnight at 4°C. The super- natant fraction (non-polysaccharide fraction) was con- centrated in a rotary evaporator at 50°C and adjusted to the same volume of the polysaccharide fraction with distilled water. The sugar content in each fraction was measured by a phenol-sulphuric acid method (Dubois et al 1962).

Assay for DNA synthesis by spleen cells

DNA synthesis by splenocytes was assayed using a slight modification of an earlier method (Okai et al 1985). Splenocytes (5 x 10') of C3H/HeJ mice (8 weeks old, Seiwa Experimental Animal Lab, Ohita, Japan) were suspended in 100 p1 of RPMI 1640 medium (Nissui Seiyaku Co, Tokyo, Japan) containing 10 pl foetal bovine serum (FBS, Gibco, New York, USA). To this suspension 25 pl of test solution was added and the mixture made up to final volume of 200 pl with RPMI 1640 medium containing 10% FBS. After cell culture for 2 days at 37°C in COJair ( 5 : 95, v/v) at 95% RH, the cells were further cultured for 18 h with 10 p1 of 0.5 pCi C3H]TdR (6 Ci mmol- ', Amersham, Buckinghamshire, UK) under the same culture conditions. The cell culture was stopped by adding 500 g litre-' trichloroacetic acid (TCA) to a final concentration of 100 g litre-' TCA and the TCA-insoluble material collected on a Whatman GF/C membrane filter. After the membrane was washed

with 50 ml litre-' TCA and ethanol, it was dried and the radioactivity of the membrane measured in a Beckman scintillation counter.

Preparation of mouse phagocytic cells

Mouse phagocytic cells were prepared by the previous method (Okai and Ishizaka 1994). Five millilitres of saline was injected into the abdominal cavity of each C3H/HeJ mouse and peritoneal cells were recovered by suction with a pasteur pipette. The cells were washed with saline, suspended with RPMI 1640 medium con- taining 50 p1 ml-' FBS, overlaid on a plastic plate and kept for 1 h at 37°C. After removing non-adherent cells by washing with the same medium, the adherent cells were treated with monoclonal anti-Thy1.2 (Olac Co, Blackhorn, UK) supplemented with guinea pig com- plement and kept for 1 h at 37°C in CO,/air (5 : 95, v/v). The adherent cells were recovered by a gentle sweeping with a rubber policeman and suspended with RPMI 1640 medium and containing 50 pl ml-' FBS. The purity of phagocytes was about 98% as judged by a phagocytic test of latex bead particles.

Assay for ingestive activity of mouse phagocytic cells

Phagocytic activity of mouse peritoneal adherent cells was assayed by an earlier method (Van Furth and Diessenlhof-Den Dulk 1980). The cells were preincubat- ed with or without polysaccharide derived from L japonica for 1 h at 37°C. After washing three times with RPMI 1640 medium, 1 x lo6 cells were suspended in 1 ml of the same medium supplemented with 100 p1 ml-' FBS, 1 x 10' formalin-treated S aureus (Cowan I Strain, Wako Pure Chemicals Co, Tokyo, Japan) in 1 ml of the same medium added and the mixture incubated for 1 h at 37°C. Then, lysostaphin (Sigma Chemical Co, St Louis, MO, USA) was added to the cell culture at a concentration of 100 U ml-' for 5 min at 37°C to lyse the extracellular bacteria. The culture well was washed with saline, air-dried, fixed with methanol for 10 min and stained with Giemsa-staining solution for 7 min. The number of bacteria incorpor- ated into the host cell was counted by a Nikon micro- scopic apparatus under 400-fold magnification.

Assay for antibody production by mouse splenocytes

The assay for antibody production was carried out by earlier method (Okai and Ishizaka 1989). Splenocytes from C3H/HeJ mice were washed twice with Hank's balanced salt solution (HBSS, Nissui Seiyaku Co, Tokyo, Japan) and the cells (2 x lo6) were suspended in 100 p1 of RPMI 1640 medium containing 10 pl FBS and cultured with 25 pl of test solution supplemented

Immunomodulating activities of Japanese edible seaweed 457

with 75 pl of RPMI 1640 medium containing 10 pl FBS in CO,/air ( 5 : 95, v/v) at 95% RH at 37°C for 4 days. The number of antibody-forming cells was determined by protein A-plaque-forming cell assay as follows; 25 p1 of 10-fold diluted protein A-coupled SRBC suspension, 25 pl splenocyte suspension, 25 p1 of a 100-fold diluted anti-mouse IgM and IgG serum (Litton Bionetics, Ken- sington, USA) and 25 pl of 10-fold diluted guinea pig complement were mixed with 200 pl of 10 g litre-' warmed-up agar (Difco) containing 0.5 g litre-' DEAE dextran (Pharmacia, Uppsala, Sweden). The mixture was placed on Petri dishes, covered with a coverglass, incubated at 37°C for 4 h and the number of plaque- forming cells (PFC) were counted (mean and SD of trip- licate assays).

Assay for interleukin-la (ILla) and tumour necrosis factor a (TNFa) released from mouse phagocytic cells

Mouse phagocytic cells ( 5 x lo5) were suspended in 1 ml of RPMI 1640 medium containing 10 pl FBS and appropriate dose of polysaccharide from L japonica was added to the cell culture medium. After incubation at 37°C for 24 h in COJair ( 5 : 95, v/v), the culture medium was recovered, frozen and kept at -80°C. The amount of ILla or TNFu in the culture medium was measured by commercial cytokine assay kit (Genzyme Co, Cambridge, MA, USA) according to the method in each kit manual. A serial dilution system of the test sample in a microtitration plate was made and com- pared with the colour development of standard assay data of control purified ILla or TNFa using a spectro- photometer for EIA (Intermed, Tokyo, Japan).

RESULTS

Detection of enhancing activity for DNA synthesis of mouse spleen cells in the hot-water-soluble extract of L japonica

As shown in Table 1, we detected an enhancing activity for DNA synthesis of splenocytes in the total extract in a dose-dependent manner. Furthermore, we separated the extract into polysaccharide and non-polysaccharide fractions and assayed the effects of both fractions on DNA synthetic activity of splenocytes. Although both fractions exhibited significant stimulatory effects on DNA synthesis, the polysaccharide fraction showed much higher activity than the non-polysaccharide frac- tion (Table 2).

Effect of polysaccharide from L japonica on ingestive activity of phagocytic cells against S aureus

We investigated the effect of the polysaccharide fraction on ingestive activity of phagocytic cells against S aureus.

TABLE 1 Effects of the extract from L japonica on DNA synthetic activ-

ity of splenocytes of C3H/HeJ mice'

Test sample C3H] TdR incorporation (CPW

Control (PBS) Dilution ratio

1 : 20 1 : 50 1 :150

379 k 26

9860 * 1204 4575 _+ 380 1271 * 165

a The hot-water-soluble extract of L japonica was prepared as described in the Materials and Methods section. The extract was successively diluted with phosphate-buffered saline (PBS) and 20 pl of each dilution was added to the cell culture system at a final volume of 200 p1 which corresponds to 10 pl, 4 p1 or 1.33 p1 of the original extract. The values in the table show the mean SD of triplicate assays.

After the cells were treated with the polysaccharide frac- tion and washed, the cells were incubated for 1 h at 37°C in the presence of formalin-treated bacteria. As shown in Fig 1, when the cells were treated with the polysaccharide at 10 p g ml-', most cells were activated to show much incorporation of bacteria compared with the control experiment (Fig l a and b). The addition of a much higher concentration of polysaccharide (50 pg ml-') caused much stronger phagocytic activity against S aureus in which considerable numbers of phagocytic cells incorporated more than 50 bacteria into each cell (Fig lc). These observations suggest that

TABLE 2 Separation of polysaccharide and nonpolysaccharide fractions

of the extract of L japonica" ~ ~

Test sample ['HI TdR incorporation ( C P M )

Control (PBS) 290 k 18 Polysaccharide fraction, dilution ratio

1 : 20 5631 413 1 : 50 3905 287

1 : 20 1870 161 1 : 50

Non-polysaccharide fraction, dilution ratio

856 k 54

After the polysaccharides of the extract was precipitated with ethanol and the supernatant (non-polysaccharide fraction) was concentrated by a rotary evaporator at 50°C. The volume of non-polysaccharide fraction was adjusted to the same volume of polysaccharide fraction with distilled water. The sugar content of polysaccharide and non- polysaccharide fractions was estimated to be about 1450 pg ml-' and 30 pg ml-', respectively. The dilution of each fraction was carried out by the same method as in Table 1. The values in the table show the mean SD of triplicate assays.

458

3 0 .

2 0 .

10

n-

r

- 45.5 f 17.0 (11462) 7 -

-7

'

n i l

Y Okai et a1

1 31.1 t 16.2 (11-1601

0 5 10 20 30 40 50 6 0 70 80 90

NUMBERS OF BACTERIA INTO CELL

Fig 1. Effect of polysaccharides from L japonica on ingestive activity of phagocytic cells against S aureus. Mouse phagocytic cells were cultured with formalin-treated S aureus for 1 h at 37°C in (a) the absence or the presence of (b) 10 pg ml-I polysaccharides or (c) 50 pg ml- polysaccharides. The distribution of phagocytic population incorporating different numbers of bacteria was illus-

trated in the figure. The value in each experiment expressed as the mean & SD.

the polysaccharides from L japonica have a possible ability to enhance the phagocytic activity of mouse immunocompetent cells against S aureus.

Effect of polysaccharide from L japonica on polyclonal antibody production in mouse spleen cells

As indicated in Table 3, when the polysaccharide (10 pg ml-') was added to the spleen cell culture, a sig- nificant stimulatory effect on polyclonal antibody (IgM and IgG) production was observed and the treatment with much higher concentrations of polysaccharide (25- 100 pg ml-l) caused much more antibody production in a dose-dependent manner. However, in another control experiment, E coli lipopolysaccharide (50 pg ml-') did not cause a significant increase of anti- body production. This result suggests that the poly- saccharide from L japonica has an adjuvant activity for antibody production of B lymphocytes of C3H/HeJ mice.

Effect of polysaccharide from L japonica on the release of ILla and TNFa released from phagocytic cells

As shown in Table 4, when the cells were treated with 10 pg ml-' polysaccharide, a significant increase in ILla concentration was observed in the cell culture medium compared with that of the control experiment. Furthermore, the addition with higher concentrations of polysaccharide (25-100 pg m1-I) caused much more

stimulation for the release of ILla from the cells. However, E coli lipopolysaccharide (50 pg ml- ') did not show the significant stimulation for the release of ILla.

As indicated in Table 5, the polysaccharide-treated cells released a greater amount of TNFa than that in the control experiment. Although a high concentration of E coli lipopolysaccharide (50 pg ml-I) showed weak enhancing activity for the release of TNFa, the similar

TABLE 3 Effect of polysaccharide from L japonica on polyclonal anti-

body production in C3HIHeJ mice splenocytes"

Test sample Antibody production (PFC per 106 cells)

I g M IgG

Control (PBS) 168 f 14 203 & 18 Polysaccharide (pg ml- I )

10 265 f 20 309 & 27 25 350 f 25 495 f 32 1 00 695 f 80 821 59

50 187 & 10 225 f 15 E coli lipopolysaccharide (pg ml - I )

(1 Appropriate doses of each test sample were added into the assay system for the antibody production in splenocytes of C3H/HeJ mice as described in the Materials and Methods section. The values in the table show the mean SD of tripli- cate assays in each experiment.

Immunomodulating activities of Japanese edible seaweed 459

TABLE 4 Effect of polysaccharide from L japonica on interleukin la rel-

eased from phagocytic cells"

Test sample I L la concentration ($9 m1-9

~~~

Control (PBS) 6 + 1 Polysaccharide (pg ml- ')

10 28 2 5 25 85 _+ 13 100 256 + 30

50 11 f 3 E coli lipopolysaccharide (pg m1-I)

Different amounts of test samples were added to the culture medium of mouse phagocytic cells as described in the Methods section. After the culture for 24 h, the culture medium of each experiment was recovered and IL-la concen- tration in the medium was assayed. The value is expressed as the mean and SD of triplicate assays in each experiment.

concentrations of polysaccharide from L japonica (25- 100 pg m1-I) caused much stronger activity for the release of TNFa compared with that of lipopolysaccha- ride. These results indicate that polysaccharide from L japonica has a possible enhancing activity for the release of cytokines such as ILla and TNFa from mouse phagocytic cells.

DISCUSSION

The results suggest that the hot-water-soluble extract of L japonica contains immunomodulating activity and that its polysaccharide fraction has much higher activity than that of the non-polysaccharide fraction.

TABLE 5 Effect of polysaccharide from L japonica on tumour necrosis

factor a released from phagocytic cells"

Test sample TNFa concentration bg mj-

~~ ~ ~~ ~~

Control (PBS) 10f 1 Polysaccharide (pg ml- I )

10 42 + 6 25 91 f 10 100 201 & 18

50 16 f 2 E coli lipopolysaccharide (pg ml- ')

' Different doses of test samples were added to the phagocytic cell culture as shown in the Methods section. The culture medium of each experiment was recovered and TNFa concen- tration was measured as described in the Methods section. The value in the table shows the mean +- SD of triplicate assays in each experiment.

To differentiate the true immunological activity from the endotoxin-induced activity we used the immuno- competent cells of the endotoxin-nonresponder strain of mice. Thus, the major immunomodulating activities in this fraction are associated with polysaccharides them- selves, but not with contaminating endotoxins by the following reasons.

As shown in Table 2, the polysaccharide fraction from L japonica showed a remarkable C3H]TdR incorp- oration of spleen cells (5631 CPM) at about 75 pg rnl-', but similar concentrations of E coli lipo- polysaccharide (50 pg m1-l) caused much lower DNA synthetic activities (725 2 31 CPM).

The experimental system for measuring antibody pro- duction was designed to remove the endotoxin-induced artificial activity. In this system, previously reported by one of the authors, the spleen cells of endotoxin- nonresponder mice are completely non-responsive to the endotoxin at a high cell density and did not show any significant antibody production (Ishizaka 1983). In the present study, the polysaccharides from L japonica caused a considerable stimulation on polyclonal anti- body production at a high density of C3H/HeJ sple- nocytes, but high concentration of E coIi lipopolysaccharide did not show significant antibody production (Table 3). Furthermore, as shown in Tables 4 and 5, the polysaccharide fraction exhibited a stimu- latory effect on the release of cytokines from phagocytic cells of C3H/HeJ mice, but a relatively high concentra- tion of lipopolysaccharide (50 pg ml-') did not cause a significant stimulation compared with those of the polysaccharide-treated cells.

These results suggest that the immunomodulating activities in the polysaccharide fraction of L japonica are closely associated with the polysaccharides them- selves, but not with contaminating endotoxins.

Previously, other investigators showed that the oral administration with the powder of edible seaweeds caused a significant decrease in the incidence of chemi- cally induced carcinogenesis in in uiuo animal experi- ment (Yamamoto and Maruyama 1985). Although the authors discussed the anti-tumour effect of poly- saccharide, the details of the immunological mechanism for the anti-tumour effect have not been analysed. The findings in the present paper offer a possible explana- tion for the anti-tumour effect by seaweeds. On the other hand, the recent study of the immunological regu- lation in epithelial mucosa of the intestinal tract has indicated that M cells in the Peyer's patches capture microorganisms or macromolecules which were trans- ported to the enfolded immunocompetent cells such as lymphocytes and macrophages. The stimulated immu- nocompetent cells migrate through lymphatics to mesentric lymph nodes and via the thoracic duct to the general circulation which affects the immune responses in the whole body (Kato and Owen, 1994). The poly- saccharides from L japonica might cause the in vivo

460 Y Okai et a1

antitumor effect by M cell-dependent pathway in the immunological regulatory networks.

Recently, significant antimutagenic activities were observed in the hot-water-soluble extracts from edible brown algae such as L japonica, Undaria pinnat$da and Hijikia fusiforme which showed suppressive effects on umu C gene expression in SOS response of Salmonella typhimurium (TA 1535/pSK 1002) induced by carcino- genic substances (Okai et a1 1993; Okai and Higashi- Okai 1994). The authors also found that organic-solvent-soluble extracts of edible seaweeds caused suppressive effects on tumour promotor-induced biochemical activation in in uitro experiments (Okai et a1 1994). Although the authors cannot determine which activity in seaweed extracts plays a dominant role in protection against carcinogenesis, the additive or syn- ergestic effects of these activities in the seaweed extracts seem to contribute the protection against actual carci- nogenesis because there are various protective pathways at various steps in carcinogenesis (Bertram et a1 1987).

Previously, anti-tumour polysaccharide was found in another brown seaweed Sargassum fulvellum which was characterised to be a sodium alginate containing man- nuronic and gluronic acids (Fujihara et a1 1984). However, no analysis for immunological activities was done. On the other hand, we could not detect significant amounts of mannuronic and gluronic acids in the acid- hydrolysed product of the polysaccharides from L japonica. The difference between these results seems to derive from the different extraction methods for the polysaccharides. The authors in the previous paper extracted the polysaccharides by boiling for 4 h under an alkaline condition to solubilise sodium alginate, while we carried out a very mild extraction in which hot water was added and the mixture held for 30 min. Poss- ibly, the immunomodulating polysaccharides in the present paper are extracted selectively from other com- ponents of polysaccharide such as sodium alginate.

REFERENCES

Bertram J S, Kolonel, L N, Meysken F L 1987 Rationale and strategies for chemoprevention of cancers in humans. Cancer Res 47 3012-3031.

Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F 1962 Colorimetric method for determination of sugars and related substances. Anal Chem 28 350-356.

Fujihara M, Iijima N, Yamamoto I, Nagumo T 1984 Purifi- cation and chemical and physical characterization of an antitumor polysaccharide from the brown seaweed Sargass- um fuvellum. Carbohydr Res 125 97-106.

Ishizaka S 1983 Modification of lipopolysaccharide non- responder cells in low cell density culture. Immunol Lett 6

Kato T, Owen R L 1994 M cell functions. In: Handbook of Mucosal Immunology, ed Ogra P L. Academic Press, New York, USA, pp 20-24.

Maeda M, Nisizawa K 1968 Fine structure of laminarin of Eisenia bicycis. J Biochem 63 199-206.

Okai Y, Higashi-Okai K 1994 Identification of antimutagenic activities in the extract of an edible brown alga, Hijikiafusi- forme (Hijiki) by umu gene expression system in Salmonella typhimurium (TA 1535/pSK 1002). J Sci Food: Agric 66

Okai Y, Ishizaka S 1989 Spontaneous release of B lympho- cytes enhancing factors for growth and differentiation by Y H-1 cells in serum-free culture media-Identification of interleukin 1 activities. Zool Sci 6 523-532.

Okai Y, Ishizaka S 1994 A possible immunomodulating activ- i ty of Arbekacin (ABK), A newly synthesized antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). Int J lmmunopharmacoll6 321-327.

Okai Y, Higashi-Okai K, Nakamura S 1993 Identification of heterogenous antimutagenic activities in the extract of edible brown seaweeds, Laminaria japonica (Makonbu) and Undaria pinnatiJda (Wakame) by umu gene expression system in Salmonella typhimurium (TA 1535/pSK 1002). Mutation Res 302 63-70.

Okai Y, Higashi-Okai K, Nakamura S, Yano Y, Otani S 1994 Suppressive effects of the extracts of Japanese edible sea- weeds on mutagen-induced umu C gene expression in Sal- monella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornuthine decarboxylase induction in BALB/c 3T3 fibroblast cells. Cancer Lett 87 25-32.

Okai Y, Gotoh S, Yamashita U 1985 Thymocyte-activating factors from SV40-transformed human embryo fibroblasts. Immunol Lett 9 153-159.

Statistical Bureau, Management and Coordination Agency Japan (1991) In: Annual Report on the Family Income and Expenditure Survey in Japan. Japanese Statistics Associ- ation, Tokyo, Japan, pp 298-307.

Statistical Information Department, Ministry’s Secretariat, Ministry of Health and Welfare Japan 1988. In: The Major Causes of Death and the Corrected Mortality in Japan. Health and Welfare Statics Association Japan, Tokyo, Japan, pp 20-25.

Van Furth R, Diessenlhof-Den Dulk M M C 1980 Method to prove ingestion of particles by macrophages with light microscopy. Scand J Immunoll2 265-269,

Willet W C 1994 Diet and health: what should we eat? Science 264 532-537.

Yamamoto I, Maruyama H 1985 Effect of dietary seaweed preparation on 1,2-dimethylhydrazine-induced intestinal carcinogenesis in rats. Cancer Lett 26 241-25 1.

343-350.

103-1 10.