Medium-term bioassays for carcinogenicity of chemical mixtures.

Carcinogenic effects of chemical mixtures were examined with a medium-term liver bioassay for carcinogens or a multiorgan medium-term bioassay using male F344 rats. In the medium-term liver bioassay, rats were initially treated with diethylnitrosamine (DEN) at 200 mg/kg body weight, i.p.; after 2 weeks they received chemical mixtures such as 10 different heterocyclic amines at one-tenth or one-hundredth the dose levels used in carcinogenicity studies and the mixtures of 20 different pesticides, each at acceptable daily intake (ADI) levels or a mixture of 100 times ADI levels. All animals were subjected to two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. The number and areas of glutathione S-transferase placental form (GST-P) positive foci (preneoplastic lesions in the liver) were compared between respective groups. When 10 heterocyclic amines were mixed in the diet at one-tenth dose level, clear synergism was observed, but no combined effects were evident with the one-hundredth dose levels. In the pesticide experiment, treatment of rats with the 20-pesticide mixture at the ADI dose level did not enhance GST-P-positive foci. In contrast, a mixture of 100 times the ADI significantly increased those values. In a multiorgan bioassay of 28 weeks, mixtures of 40 high-volume compounds and 20 pesticides (suspected carcinogens) added together at their respective ADI levels did not enhance carcinogenesis in any organs initiated by five different carcinogens (DEN, N-methylnitrosourea, dimethylhydrazine, N-butyl-N-(4-hydroxybutyl)nitrosamine, and dihydroxy-di-n-propylnitrosamine) in combination. The combination effect of low dietary levels of five antioxidants, butylated hydroxyanisole, caffeic acid, sesamol, 4-methoxyphenol, and catechol, were also examined using the multiorgan bioassay. The incidence of forestomach papillomas was significantly increased only in the combination group and the results indicate that combination of the five antioxidants can exert additive/synergistic effects on tumorigenesis in the multiorgan bioassay. These results indicate that chemical mixtures at very low doses did not enhance preneoplastic lesions synergistically but the mixtures at certain doses show synergism in the target organ. The medium-term bioassays are particularly useful tools for this purpose.

Our environment contains a great variety of carcinogenic factors including naturally occurring and synthetic carcinogens, radiation, and viruses, all of which have been speculated to play major roles in the etiology of human cancers (1). Because humans are exposed concurrently or sequentially to a large variety of environmental carcinogens at only very low individual doses over their lifetime, a strong possibility exists that This paper is based on a presentation at the Conference on Current Issues on Chemical Mixtures held [11][12][13] August 1997 in Fort Collins, Colorado. Manuscript received at EHP 17 February 1998; accepted 15 July 1998.
Address correspondence to N. Ito, Nagoya City University, 1  agents may act in combination to induce cancers. Therefore, in addition to detecting carcinogens in our environment, examination of low-dose combination effects of such agents is an important area for research to evaluate human cancer risk. Many combinations and scenarios are possible with exposure to multiple chemicals, depending on the mode of application and whether administration of carcinogenic and/or noncarcinogenic agents is simultaneous or sequential. The two-stage carcinogenesis hypothesis, now generally accepted as a basic theory of carcinogenesis, assumes sequential exposure to initiating agents and modifiers acting to promote the process of neoplasia.
To bridge the disadvantages of long-term carcinogenicity tests and in vitro mutagenicity assays, medium-term bioassays using preneoplastic lesions as end point markers have been proposed. A mediumterm liver bioassay for carcinogens and a multiorgan bioassay can be used for detecting the effect of chemical mixtures at low dose levels, as well as detection of the carcinogenic potential of individual test chemicals.

Materials and Methods
Experiment I Medium-Term Liver Bioassay for Carcinogens. Male F344 rats (group 1) were given a single ip injection of diethylnitrosamine (DEN) (200 mg/kg body weight) to initiate hepatocarcinogenesis; after a 2-week recovery period they received a test compound(s). The experimental protocol for the medium-term bioassay is shown in Figure 1. Groups 2 and 3 were given DEN alone and saline control test compound(s), respectively. All animals were subjected to two-thirds partial hepatectomy at week 3 and sacrificed at week 8. Liver splices were fixed in icecold acetone, embedded in paraffin, then immunohistochemically stained for glutathione S-transferase placental form (GST-P). Numbers and areas of GST-P-positive hepatic cell foci larger than 0.2 mm in diameter as well as the total areas of the liver sections examined were measured using a color video image processor. Quantitative values for GST-P positive foci were compared between the chemical-treated group and the control group. We consider this model eminently suitable for evaluation of combined effects of several carcinogenic Environmental Health Perspectives * Vol 106, Supplement 6 * December 998 ITO ET AL. agents (2). Statistical analysis of differences between means was carried out using the Student's t-test or the Welch's t-test after application of the preliminary F-test for equal variance for each pair. For proportion data, the Fisher exact probability test was used. To determine whether the combined treatments acted additively or synergistically, one-tail analysis by the modified Colton's z-test was carried out (3,4). Experiment I-A Low-Level Exposure to a Mixture of Heterocyclic Amines. Heterocyclic amines produced in foods under normal cooking conditions are highly mutagenic and induce carcinogenicity in rodents (5). Humans are chronically exposed to mixtures of these agents simultaneously at low dose levels in the diet (6). Therefore, potential synergism  Table 1. All pesticides examined were organophosphates, except for endosulfan. The animals were initially given a single ip injection of DEN. After a 2-week recovery period, the rats received the pesticides either at the ADI (mixture one, group 1-A) or at 100 times higher doses (group 1-B) or were maintained on the basal diet throughout the experiment (group 2). Groups 3-A and 3-B were injected with saline and then fed the mixture one at the ADI and 100 times the ADI, respectively. All animals were subjected to two-thirds partial hepatectomy at week 3 and sacrificed at week 8.
Liver slices were stained for GST-P.
Numbers and areas of GST-P-positive hepatic cell foci larger than 0.2 mm in diameter and the total areas of liver sections examined were measured using a video image processor. insecticide. 1ADI levels were provided by the Ministry of Health and Welfare, Japan (9), except for dimethoate, endosulfan, and methidathion, for which data were cited from FAO/WHO (9). bYear refers to the date of the study as published by FAO/WHO (9). cLower than the ADI levels from FAO/WHO (9).
Environmental Health Perspectives * Vol 106, Supplement 6 * December 1998 1 332 Experiment II Multiorgan Bioassay. To detect carcinogenic or modifying potentials of test chemicals in multiple organs as well as the liver for the medium-term period, we also developed a medium-term multiorgan bioassay (DMBDD model) (4,10). Figure 2 shows the experimental protocol of the multiorgan medium-term bioassay. As initiation, five known potent carcinogens were given in combination within the first 4 weeks: a single ip injection of DEN at a dose of 100 mg/kg body weight at the start of the experiment; four ip injections of N-methyl-N-nitrosourea (MNU) at a dose of 20 mg/kg body weight on days 2, 5, 8, and 11, and four sc injections of 1,2-dimethylhydrazine (DMH) at a dose of 40 mg/kg body weight on days 14, 17, 20, and 23, 500 mg/1 N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in the drinking water during weeks 1 and 2, and 1000 mg/1 2,2'dihydroxy-di-n-propylnitrosamine (DHPN) in the drinking water during weeks 3 and 4. After this DMBDD treatment, rats received test chemicals for 24 weeks. Noninitiation controls were injected ip with saline and subcutaneously with corn oil and then given pesticide(s). At week 28 of the experiment, all surviving animals were sacrificed and completely autopsied. Livers were analyzed for GST-P-positive foci as described in Experiment I. The main organs and any macroscopic lesions were removed and fixed in formalin. The routinely prepared hematoxylin and eosin sections were examined for neoplastic and preneoplastic lesions. Possible modifying effects of these pesticide mixtures on tumorigenesis were investigated using the DMBDD model. After the DMBDD treatment, groups of rats received one of the pesticide mixtures (group 1 received mixture one and group 2

Results
Experiment I-A When 10 heterocyclic amines were mixed in the diet at the one-tenth dose level, clear synergism was observed, but no combined effects were evident with the one-hundredth dose levels ( Table 2) (3). The fact that synergism clearly was observed at relatively low dose levels is important because mixtures of these heterocyclic amines may be generated in cooked food. Although individual compounds might be without obvious effects on carcinogenicity, they may present a hazard risk in combination at the levels present in food.

Experiment I-B
Data on the numbers and areas of GST-Ppositive foci per unit area of liver section with and without DEN initiation are compared in Table 3. The number of GST-P-positive foci in group 1-A was 3.36 ± 1.29/cm2 and the area of foci was  None-ADI mixture 10 0 0 5 None 100xADI mixture 9 0 0 *`**Significantly different from the relevant group value at p< 0.05 and p< 0.01, respectively.
0.29 ± 0.15 mm2/cm2. The levels were essentially the same as those in the control group (3.50 ± 1.29/cm2 and 0.28 ± 0.13 mm2/cm2). However, the values obtained in the 100 times higher ADI mixture group (group 1-B) (4.51 ± 1.64/cm2 and 0.44 ± 0.20 mm2/cm2) were both significantly higher than the control values. Without the DEN initiation, neither of the treatment schedules induced GST-Ppositive liver cell foci larger than 0.2 mm in diameter (groups 3-A and 3-B). Experiment II-A In the liver, development of GST-P-positive foci was increased by captafol but not modulated by the mixtures (Figure 3). In the other organs, captafol showed promotion effects in the thyroid, whereas the pesticide mixtures did not influence the neoplastic development in any organ. No neoplastic and preneoplastic lesions were observed in noninitiated groups (groups 3-A to 3-C) ( Table 4). Experiment II-B In the carcinogenicity study, slightly increased incidences of forestomach papillomas were found in the sesamol-(15.8%), caffeic acid-(14.8%), catechol-(3%), and 4-MP-(11.5%) treated groups as compared to the basal diet group (0%); a significant increase was observed with the five antioxidants in combination (42.9%, p< 0.001) ( Table 5). In the medium-term multiorgan bioassay, incidences of forestomach papillomas and/or carcinomas were increased in  each high-dose group, but additive or synergistic effects were not found in the combination group. In the low-dose group, the incidence of forestomach papillomas was significantly increased only in the combination group. With regard to other organs, the incidence of colon tumors was significantly decreased only in the highdose combination group. The results indicate that even at relatively low dose levels, phenolic compounds can exert an additive/ synergistic effect on carcinogenesis (12).

Discussion
In the liver model, the ADI mixture of organophosphorus pesticides exerted no effects on development of liver preneoplastic foci initiated by DEN, although the 100 times higher dose demonstrated lesion-promoting potential (8,13,14). In the multiorgan model, the ADI mixtures of 40 (mixture two) or 20 (mixture three) pesticides demonstrated no tumor-promoting potential in any organ or tissue (13,14). Captafol, on the other hand, exerted apparent tumor-promoting effects in the liver, thyroid, and kidney, although the dose level was not comparable to the mixtures. The protocol has been developed in our laboratory over the last 15 years (10). Quantitative analysis of GST-P-positive foci has been established. The multiorgan method has been developed to supplement the liver model and is also a useful method for rapid detection of carcinogens at the whole-body level (4,10).
With a safety factor approach, acceptable exposure levels such as ADIs are usually determined by dividing the no observed effect level from laboratory-based chronic toxicity tests by an appropriately chosen safety factor. The safety factor used for ADI by the Japanese Ministry of Health and Welfare and the FAO/WHO is usually 100, but WHO expert committees have used figures ranging from 10 to 2000 (15). Our experimental results indicate that this procedure is indeed appropriate and acceptable for risk evaluation at present. Furthermore, the chance of exposure to so many pesticides (20 or 40 chemicals) in concert might in practice be low (16).
Because most human cancers may be caused by trace environmental factors, it is of increasing importance that combined effects of chemicals at relatively low doses be examined. The medium-term bioassays used in these studies are particularly useful methods for this matter.