Effects of low dose mixtures of four N-nitroso compounds on hepatic foci development in the rat

CancerLetters 106( 1996)263-269

Effects of low dose mixtures of four N-n&rosa compounds on hepatic foci development in the rat Mitsuru Futakuchi*, William Lijinskyl, Ryohei Hasegawa, Masao Hirose, Nobuyuki Ito, Tomoyuki Shirai First

Department

cfPathology,

Nagoya

City

University

Medical

School,

I-Kawa~mi,

Mi.&n-cho,

Mizuho-ku,

.&gnyo

$67,

Jqxuz

Received 8 May 1996;revisionreceived24May 1996;accepted 24May 1996

Abstract Potential synergismbetweenfour N-nitroso compounds(nitrosomorpholine,nitrosodimethylamine, nitrosodiethanolamine,nitroso-oxazolidine)in rat liver carcinogenesis wasexaminedin the medium-term bioassay.MageF344 ratswere initially given diethylnitrosamine(DEN, 200mg/kg, ip) andbeginning2 weekslater receivedtest chemicalsfor 6 weeksindividually at a full or l/4 doseof that provento becarcinogenicindividually or in combination.All animalswaresubjectedto partial hepatectomyat week 3 and killed at week 8. Induction of immunohistochemically-demonshated glutathioneStransferase placentalform (GSTP) positive foci wasevaluated.The numbersandsize of GST-Ppositivefoci were significantly higherthan the control levelsby the treatmentwith eachnitrosamineat full (l/l) andone quarterdoses(114),excepting nitrosodiethanolamine andby combinationof the four chemicalsat l/4 and l/16. Becausethedose-response curveswere considered non-linearfor mostnitrosamines, synergisticeffectswerenot apparentfor the l/4 mixture. In~~st~~~, however, the valuesfor ratstreatedwith thesefour chemicalsin combinationat the l/4 doselevel werealmostthe sameasthe average of four individual treatmentsat the full dose,andthosefor the l/16 dosemixture werealmostthe sameasthe averageof l/4 individualtreatmentgroups.Theseresultsindicatethat thesenitrosamines workedadditively,ratherthan synergistically,in rat liver carcinogenesis. Keywords: Nitrosamine;Carcinogenesis; Liver; Rat

1. Introduction Epidemiological studies have suggestedthat most human cancersare causedby exposure of individuals to environmental carcinogenic agents and interest in a practical approach to cancer prevention is therefore * Corresponding author. ’ Presentad&~: 11398High Hay Drive, Columbia,MD 21044,USA.

increasing. Among theseenvironmentai factors, various types of carcinogenic agentshave been identified in our environments. We selected four nitrosamines found in our environment and to which we might be exposedtogether at low doses.Ni~s~~~~~~~ine (NDMA) is the most widely distributed carcinogenic nitrosamine (in food, pesticide formulations, tobacco smoke and industrial environments) [ 11.Nitrosomorpholine (NMOR) is an important contaminant of rubber and is present at significant concentrations in

0304-3835/96/$12.00 0 1996ElsevierScience IrelandLtd. All rightsreserved P/I SO304-3835(96)043261

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factories where rubber is made or processed and is also present in some smokeless tobaccos [2]. Nitrosooxazolidine (NOX) is a close analog of NMOR and has been found in cutting and grinding fluids used in metal machine shops. N-Nitrosodiethanolamine (NDELA) is considered one of the N-nitroso compounds to which human exposure is greatest. It has been found under specific workplace conditions [35] and in cosmetics [6], pesticides, tobacco, and tobacco smoke [7]. The importance of considering chemical mixture exposure in dealing with environmental issues has recently become better recognized. Humans are clearly exposed concomitantly or sequentially to several environmental carcinogens at low doses under normal conditions [8,9]. Long-term animal experiments focusing on this issue are practically rather difficult, since they require large animal numbers and the evaluation of combined effects is complicated. As one approach to the present problem, we have conducted the present experiments using a so-called medium-term liver bioassay for carcinogens [ 10,111. Based on the two-step carcinogenesis hypothesis, the simplest model allows rapid examination of liver carcinogenic potential of chemicals using rat placental glutathione S-transferase (GST-P) positive hepatic foci as the endpoint marker lesions [ 10,111. Hasegawa et al. [ 121 and Ito et al. [ 131 reported synergistic effects of five or ten heterocyclic amines using this medium term bioassay. The aims of our study were (1) to evaluate the carcinogenic potential for each chemical and a doseresponse relationship and (2) whether they act additively or synergistically when administrated together, as determined by the medium-term liver bioassay. 2. Materials

and methods

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106 (1996)

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lected on the same basis as for previous studies [14, 151. Each compound was incorporated at two concentrations in drinking water: NDMA 20 and 5 mg/l; NMOR 40 and 10 mg/l; NOX 36 and 9 mg per liter; and NDELA 100 and 25 mg/ 1. We also examined the effect of all four carcinogens at higher (l/4) and lower (l/16) mixture concentrations. The drinking water solutions were dispensed at a rate of 20 ml/day to each rat (80 ml for a cage of four rats) on 5 days of each week (all of which was consumed), followed by tap water ad libitum for the remaining 2 days of each week. 2.2. Animals and treatments A total of 165 male F344 rats (Charles River Japan Inc., Atsugi, Japan), 6 weeks old at the start of the experiment, was maintained on basal diet (Oriental MF; Oriental Yeast Co. Ltd, Tokyo, Japan) ad libitum and housed in plastic cages in an airconditioned room at 24 + 2°C and 55 + 5% humidity with a 12: 12 h light/dark cycle. Fig. 1 shows the basic protocol of the mediumterm liver bioassay [ 10,l 1,161. In the present experiment, the animals were divided into 11 groups of 15 rats each (see Table 1). All rats were given DEN (200 mg/kg body weight, i.p.) at the start of the experiment and beginning 2 weeks after that received one of the four nitroso compounds at full or l/4 dose or a combination of all four chemicals at l/4 or l/16 of each full dose. Animals in group 11 received no nitroso compounds as control. All rats were subjected

1

2.1. Chemicals and dose selection

2

Diethylnitrosamine (DEN) was obtained from Tokyo Chemical Co., Tokyo. NDMA, NMOR, NOX, and NDELA were prepared in our laboratory. The purities of these nitroso compounds were confirmed by high-pressure liquid chromatography, mass spectrometry, and NMR spectrometry. Doses used in the present combination study were l/l, l/4 and l/16 of the doses proven to be carcinogenic (full doses), se-

3

V

s

Fig. 1. Experimental design. As test chemicals, NDMA, NMOR, NOX, NDELA and their combination were given at two concentrations in drinking water as described in Section 2.

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et al. /Cancer

to two-thirds partial hepatectomy at the end of week 3 and sacrificed at week 8. Immediately upon killing, the livers were excised and weighed. Slices from the right anterior and caudate lobes were fixed in icecold acetone for immunohistochemical examination of GST-P expression as previously described [ 171. 2.3. GST-P positive,foci analysis In the present experiment, GST-P positive foci larger than 0.2 mm in diameter were counted [ 131, using a video image processor (VIP-21C, Olympus Co., Tokyo) and expressed as numbers and areas (mm*) per unit area of liver (cm*). Statistical analysis was carried out using the Student’s t-test and Welch’s t-test in combination with the F-test for equal variance. Statistical analysis was performed to determine whether the combined treatments acted additively or synergistically as previously suggested [ 12,131. Table

Latter,7 106 (1996)

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165

3. Results Final body and liver weight data are summarized in Table 1. Body weights of rats treated with l/4 or 1/I 6 combination and control were 274 +- I 1, 284 +14, and 281 + 15 g, respectively, and no significant differences were seen between them. In the single chemical treated groups. a significant decrease (P < 0.05) was seen in the body weights of rats treated with NMOR at the full dose; however, no significant decrease or increase were seen in the body weights of the rats treated with other nitrosamines at both full and quarter doses. Dose dependency in body weight gain were seen in each nitrosamine, and combination group. In contrast to body weights, relative liver weights were larger than that of control group except for NDELA treatment groups. In this treatment group, no apparent dose responses were seen in relative liver weight. Significant decreases in relative kidney

I

Final body and organ weights Treatment

Dose

No. of rats

Body weight(g)

Relative

organ weights -

Liver DEN+ NMOR NDELA NOX NDMA NMOR NDELA NOX NDMA l/4 combination l/16 combination DEN alone NMOR NDELA NOX NDMA NMOR NDELA NOX NDMA l/4 combination I /I 6 combination

F F F F 114 l/4 114 114

F F F F l/4 l/4 114 l/4

15 14 14 14 14 14 14 11 13 14 14 7 7

271 -t 278 2 273 ‘2742 279r 285 t 282 t 2762 274 + 284 rt 281 + 297 c 302 + 293 r 283 f 296 f 304 * 297 i: 306 + 302 + 29926

1 ii’ 13 13 I? I:! 10 14 IO II 14 15 13 11 12 13 7 10 15 5 9

Data are mean values + SD. a-b*cSignificantly different from the DEN alone value at aP < 0.05, bP < 0.01, ‘P < 0.001.

3.44*o.14c 3.16+0.11 3.58 f 0.14’ 3.30 z+z0. IX” 3.36+0.13’ 3.22 + 0.14 3.49 f o.22c 3.35 2 0.22” 3.34 -t 0.16b 3.36 2 0.10’ 3..6~Oo.l? 3.27 + 0.13 3.34 + 0.10 3.72 -e 0.17 3.32+0.14 3.4OkO.12 3.38 + 0.09 3.4420 17 3.46 + 0.16 3.61 zt 0.08 3.59 + 0.14

i%:) Kidneys

266

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weight were seen in NDELA at both dose and NMOR at a quarter dose. No significant differences were seen in other groups compared to the control group. The body weights of the rats without DEN treatment were 283-306 g, and relative liver weight and kidney weight were 3.27-3.72 g and 0.62-0.64 g, respectively. 3.1. GST-P positive foci Data for numbers and areas of GST-P positive foci per unit area of liver section are illustrated in Fig. 2 and Table 2. Numbers (No./cm2) of GST-P foci were between 5.8 and 21.4 (control level, 5.9) and areas (mm2/cm2) were between 0.4 and 1.2 (control level was 0.4). Except for the NDELA group at a full dose, in all other individual chemical treatment groups at the full and quarter doses and in the l/4 and l/16 combined group, the levels of GST-P positive foci were higher than the control group. The values of the number and the area of foci in l/4 combined treatment were 12.7/cm2 and 1.1 mm2/cm2. The number

Fig. 2. Quantitative analysis of GST-P positive foci. Individual values are significantly different from the DEN alone value at *P < 0.05, **P < 0.01, ***P < 0.001. In the comparison of the value of 114 combination and sum of the effects of each individual treatment at a quarter dose, no significant increase was seen between them (heteroadditive analysis). In the comparison of the average values of each individual treatment at full dose and the value of combination treatment at a quarter dose, also no significant increase was observed between them (isoadditive analysis). Additionally, in comparison of the average values of each individual treatment at a quarter dose and the value of combination treatment at 1116 dose, also no significant increase was observed between them.

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Table 2 Quantitative

analysis

Treatment

l/l -NMOR I/l-NDELA l/l-NOX l/l-NDMA l/4-NMOR l/4-NDELA l/4-NOX l/4-NDMA l/4 combination l/16 combination DEN alone l/l -average I/4-average

of GST-P positive No of animal

15 14 14 14 14 14 14 11 13 14 14

Data are mean values + SD. b~cSignificantly different from CP < 0.001.

GST-P

foci positive

foci

Number (no./cm*)

Area (mm2/cm2)

21.36 + 3.74’ 5.81 f 1.83 12.87 i 2.31’ 10.15 2 1.89’ 9.69 + 2.63’ 7.74 k 1.57h 8.25 i 2.20c 7.59 + 2.01h 12.7 1 -+ 2.57c 8.45 + 2.48b 5.87 i 1.44 12.75 f 6.27 8.36” 2.30

2.02 + 0.60’ 0.42 f 0.19 1.15 io.2sc 0.77 k 0.24’ 0.68 k 0.23’ 0.62 f 0.22b 0.60 f 0.27b 0.58 k 0.24b 1.10~o.31c 0.74 + 0.36b 0.36 + 0.10 1.11 kO.71 0.62 + 0.24

the DEN alone value

at bP < 0.01,

and the area of GST-P foci was 8.5/cm2 and 0.7 mm2/cm2, respectively in the l/l6 combined group. The average value of four individual nitrosamine treatments at a full dose were 12.8 (number) and 1.1 (area), respectively. And the average value of those at a quarter dose were 8.4 (number) and 0.6 (area), respectively. In non-DEN-initiated groups, no foci larger than 0.2 mm in diameter were observed. The dose-responses were not linear for NDELA, NOX, and NDMA but almost linear for NMOR. The effect value of individual chemical means the value of single treatment minus the value of the control group. The sum of the effects values of four individual nitrosarnines at a quarter dose were 15.8 in number, 1.3 in area. In the comparison between the value of the effects of four individual nitrosamines at a quarter dose and the value of the combination at a quarter dose, no significant differences were observed either in number or area of GST-P positive foci. Synergistic effects of the four nitrosamines on foci development for the l/4 and l/16 combined group were not observed. However, the values for rats treated with four chemicals in combination at the l/4 dose level were almost the same as the average of four individual treatments at the full dose, and those

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for the l/16 dose mixture were almost the same as the average of l/4 individual treatment groups, indicating a simple additive effect. 4. Discussion Many human cancers may be caused by trace environmental factors such as nitrosamines, so it is important to examine combined effects of those chemicals at relatively low doses. In the present study, we selected four nitroso compounds which are environmentally important, and tested combination effects at relatively low doses. Possible synergistic effects were not seen in both l/4 and l/l6 dose mixtures of compounds. In our previous study of the combination effect of ten heterocyclic amines at low doses [ 18, 191, we used two statistical models, a heteroadditive model and an isoadditive model. In the heteroadditive model we compared the sum of effects value of ten individual treatments at l/10 dose and the value for combination treatment of ten heterocyclic amines at l/10 dose. Significantly greater effects were found in the combination group, meaning synergism was observed among ten chemicals. However, doseresponse curves were not considered in this model. Reif et al. [20] pointed out the importance of the dose-response curve in the analysis of the combined effects. They described that in the ordinary heteroadditive methods the combined effect could be evaluated whether it was synergistic or not. In this heteroadditive model, the consideration of the doseresponse curves is lacking. On the other hand, in the isoadditive model, we compared the average value of individual treatments of l/l dose and the value of ten heterocyclic amines and the combination treatment at l/10 dose. In this comparison, a significant increase was not shown in the combination group, so we concluded that this combination effect was not synergistic in the isoadditive model. Indeed, we observed the value for combination treatment was much higher than the control level. However, from these two statistical models in combinations of heterocyclic amines at low dose, we concluded that the combination effect showed not true but apparent synergism, meaning isoadditivity. This shows the importance of careful judgement. In the present experiment, we compared the value of 114 combination and sum of the effects of each

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261

individual treatment at a quarter dose in Fig. 2. No significant increase was seen between them. Moreover, in the comparison of the average values of each individual treatment at full dose and the value of combination treatment at a quarter dose, no significant increase was also observed between them (Fig. 2). Similarly, in comparing the average values of each individual treatment at a quarter dose and the value of combination treatment at l/16 dose, also no significant increase was observed between them (Fig. 2). These results lead us to the conclusion that the combination effects of these four nitrosamines were not synergistic but &additive. One of the reasons why no synergistic effect was observed even in the heterodditive model is that the low dose of nitrosamines used in this experiment showed strong effects. One of the reasons that isoadditivity was observed in this study, is that each individual nitrosamine may have a similar metabolic pathway. Generally speaking, a combination effect may be caused by various biological activities of test chemicals, mainly depending on whether the agents have the same or different sites of primary action (similar or dissimilar). In independent action (dissimilar and non-inreractivej, the result of combined treatment may be simply additive. However, only the rats treated with NDELA at both full and quarter doses in this experiment showed no increase in relative liver weight and no remarkable developments of GST-P foci. These observations are in line with negative results in several routine mutagenicity tests using the Ames protocol [Zl241, although, NDELA appeared to be mutagenic in the host-mediated assay [25], and it was found that NDELA increases rates of micronuclei formation, chromosome aberrations and sister chromatid tests [26]. Mutagenic activity and potency of DNA adduct formation also differ between these four nitrosamines. NDMA is a mutagen forming DNA adducts extensively in rat liver and other organs. NMOR is a mutagen, but does not form DNA adducts as yet detectable in rat liver[l4]. NOX is a mutagen, and afso has not been shown to form DNA adducts in rat liver[l4]. NDELA is not mutagenic and forms only very small amounts of DNA adducts in rat liver [27]. In conclusion, careful analysis is needed to evaluate combination effects of carcinogens. In this study, the combination effect of four nitrosamines showed

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isoadditivity. Better statistical analysis is needed to evaluate combination effects. The present data indicates that, although individuals are usually exposed to very low doses of individual environmental chemicals, acting in concert they might be capable of inducing tumors. Acknowledgements The studies were supported in part by Grants-inAid for Cancer Research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, a Grant-in-Aid from the Ministry of Health and Welfare for the Second Term Comprehensive lo-Year Strategy for Cancer Control, and grants from the Society for Promotion of Pathology of Nagoya and the Experimental Pathological Research Association, Nagoya, Japan. W. Lijinsky was a foreign research fellow of the Foundation for Promotion of Cancer Research, Tokyo, whose support is gratefully acknowledged. References [I]

[2]

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[6]

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[9]

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