FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

18,

193-199 (1992)

Effects of Diet Type on Incidence of Spontaneous and 2-Acetylaminofluorene-Induced Liver and Bladder Tumors in BALB/c Mice Fed AIN-76A Diet versus NIH-07 Diet FLOYD R. FULLERTON,*

DAVID L. GREENMAN,*

AND THOMAS

J. BUCCI?

*National Center for Toxicological Research and tPatho1og.v Associates, Inc., NCTR. Jefferson, Arkansas 72079 Received January 4, I99 1; accepted August 30, I99 1

Effects of Diet Type on Incidence of Spontaneousand 2Acetylaminofluorene-Induced Liver and Bladder Tumors in BALB/c

Mice Fed AIN-76A Diet versus NIH-07 Diet. F.R., GREENMAN, D.L., AND Buccl,T.J.(1992). Fundam. Appl. Toxicol. 18, 193-199. FULLERTON,

Diet is a major influence on the responsesof experimental animalsto drugs,toxins, and carcinogens.Two diets usedwidely in toxicological and/or nutritional studies,and consideredto be nutritionally adequate,were comparedwith respectto their influenceon growth, body weight, lifespan,spontaneousneoplasia, and neoplastic responsesto 2-acetylaminofluorene (2-AAF). Both sexesof weanling BALB/c mice were fed either a purified diet (AIN-76A) or a nonpurified, natural ingredient diet (NIH-07), with or without 2-AAF for up to 2 years. Dosagesof 2-AAF were administeredto malesat 0, 20, 40, or 60 ppm in eachdiet and to femalesat 0, 100, 125, or 150ppm. Each group consistedof 96 mice. In most instances,malesand femalesfed purified diet (AIN-fed) gained weight more rapidly, attained higher maximum body weights, and died earlier than their nonpurified diet (NIH-fed) counterparts. 2-AAF inhibited weight gain significantly only in AIN-fed females. Thus, femalesreceiving 150 ppm 2-AAF gainedlittle more than their NIH-fed counterparts.At the dosages usedin males,2-AAF did not induce liver neoplasia but the AIN diet was clearly associatedwith a higher spontaneousfrequency of liver neoplasiathan the NIH diet. Although 2-AAF inducedliver tumors in femalesfed either diet at all dosages,a higher frequency and earlier appearanceof liver tumors amongAIN-fed femalesthan their NIH-fed coun-

terparts was apparent mainly at the lowest dosage. 2-AAF induced bladder neoplasia in both sexes.AIN-fed males were much more sensitive to this effect than NIH-fed animals; however,

diet had no impact on the induction of bladder neoplasia in females. This study dramatically shows the importance of diet selectionon the outcome of carcinogenicity studies.However, the resultssuggestthat dietary differencesmay not beconsistently found among different sexesor strains and that differencesbetweenpurified and nonpuriheddiets in the carcinogenicresponse to a carcinogen may be related to their differential effects on

body weight. o 1992 Society

of Toxicology.

Recent interest concerning the effects of diet and nutrition on cancer and the carcinogenic process has been focused in three general areas: (1) diet as the source of carcinogenic

chemicals (2) diet as a modulator of the carcinogenic process through the presence of nonnutrient compounds that alter metabolic reactions, and (3) diet as a nutritional modulator of carcinogenesis. Many dietary components increase or decrease susceptibility to chemicals (Carroll and Khor, 1975; Doll, 1979; Kritchevsky, 1986; NCI, 198 1; Newberne and Conner, 1986; Wiseburger, 1979). The alteration often cannot be predicted using knowledge of dietary effects on metabolism of the compound (Campbell and Hayes, 1974). A need for standardized animal diets in carcinogenic and toxicological research has been suggested (ILAR Committee, 1972; Ad Hoc Committee, 1977; ILAR Committee, 1978; Greenman et al., 1980; Topham and Eva, 198 1; Wise, 1981). We have previously presented results of a 2-year study with B6C3Fl mice (the hybrid stock used in the NTP bioassay program) fed either NIH-07 (the cereal-based diet used in the NTP program) or AIN-76A (a purified diet) and chronically exposed to 2-acetylaminofluorene (2-AAF), a known carcinogen (Fullerton et al., 1991). In that study, incidence of malignant liver tumors was significantly greater in all AINfed groups compared to NIH-fed groups, as was total incidence of liver tumors (malignant + benign). Similarly, the incidence of 2-AAF-induced malignant and total bladder tumors was greater in AIN-fed than in NIH-fed groups of mice administered the high doses of 2-AAF for both sexes. The purpose of the current report wasto expand data to an inbred strain of mice (BALB/c) in a similar study, with the intent of detecting major qualitative or quantitative differences in either spontaneous or induced neoplasia under the two dietary regimens when the labor intensive method of pairfeeding was not employed to assure isocaloric intake. MATERIALS

AND METHODS

Chemical. Purity ofthe 2-AAF (Aldrich Chemical Co., Milwaukee, WI) was 97.9% as determined by FID-gas chromatography. Diets and 2-AAF stability in diets. Two open-formula diets, AIN-76A (AIN), a purified diet (Bio-Serv, Inc., Frenchtown, NJ), and NIH-07 (NIH), a nonpurified, natural ingredient diet (Ziegler Brothers, Inc., Gardners, PA), were used in the study as described previously (Fullerton et al., 1991). Tests were conducted to determine the stability of 2-AAF in both diets. Levels of 25 and 200 ppm 2-AAF were mixed in each diet. Levels of 2-AAF

193

0272-0590/92$3.00 Copyright All rights

0 1992 by the Society of Toxicology. of reproduction in any form reserved.

194

FULLERTON,

GREENMAN,

AND BUCCI

TABLE 1 Body Weight Gain and Median Lifespan in BALB/c Mice Fed AIN-76A or NIH-07 Diet Body weight gain (%) Sex

Diet

F

AIN

2-AAF concn. hw-4 0 100 125

150 F

M

M

NIH

AIN

NIH

0 100

Initial weight (g) 18.9 f 18.6 f 18.7 f 18.6 +

0.9 0.8 1.0 0.9

18.7 f 1.0

4

weeks

16 weeks

9.6 + 3.7b

31.6 f

6.1b

11.5 + 3.6b 10.3 + 3.4b

31.6 f

6.5’

8.0 f 4.2'

28.5 f 26.1 +

3.6 4.8 3.4 2.9

f + k f

3.9 4.5 4.0 3.9

14.0 15.9 13.4 12.4

+ * f f

-+ f + +

3.7' 4.3' 6.0b 6.2b

54.2 55.2 52.6 54.8

f 7.6b f 7.9b k 10.4' + 9.1b

64

weeks

Median lifespan (days)

5.4' 5.2b

50.1 45.9 40.4 37.7

+ f f L!z

6.8b 7.56 6.5" 5.1bd

63.4 54.6 48.6 47.2

+ 8.8' + 10.8' t 6.1bd + 7.6bd

639' 601' 560' 507'

7.2 5.3 8.4 5.1

27.0 30.0 26.6 26.6

+ f f f

5.3 5.1 7.0 4.7

38.9 40.3 38.2 38.4

+ + k f

7.0 6.9 6.9 6.5

729 672 665 629

60.7 63.1 57.8 58.6

+ 9.9' k 11.9' + 13.1b + 9.3'

7.9' 9.8b

565' 545' 546' 448'

125 150

18.5 + 0.9 19.0 * 0.9 19.0 rf 0.8

0 100

21.9 + 1.0 21.5 f 1.0

125 150

21.9 f 1.3 22.2 f 1.1

22.9 22.8 21.3 22.4

0 100 125

21.7 f 1.0 21.6 -c 1.1

9.6 f 4.4

17.0 +

8.1

10.6 _t 6.6

19.7 f

8.7

22.0 -+ 1.2 22.3 + 1.0

9.4 AI 4.5 8.6 f 5.7

15.5 + 12.5 f

10.0

150

weeks

32

8.5

33.8 + 34.5 + 29.0 + 28.7 f

7.1

10.1 10.0 7.2

54.6 2 57.5 f 54.5 k 51.2 + 38.0 +

1l.8b 5.1b 9.0

I

41.0 + 13.5

/

39.0 + 34.9 +

f g

9.8 6.4

a The data are presented as the mean f SD. b Significantly different from NIH diet with corresponding 2-AAF dose (p < 0.01). ’ Significantly different from NIH diet with corresponding 2-AAF dose (p < 0.001). d Significantly different from corresponding control (0 ppm 2-AAF) at same time period (p < 0.01). e Significantly different from NIH diet with corresponding 2-AAF dose (p i 0.0001). f 59% survival when euthanized at 730 days. 8 9 1% survival when euthanized at 575 days.

were determined at 0,4,7, 11, and 18 days in each diet after being subjected to simulated feeding conditions and at 2, 4, 8, and 16 weeks in each diet after being subjected to simulated storage conditions. Results of these tests indicated no decrease in 2-AAF concentration when mixed in either diet at 25 ppm and subjected to 18 days of simulated feeding conditions or 16 weeks of simulated storage conditions. When mixed in the two diets at 200 ppm, 2-AAF decreased no more than 10% after 18 days under simulated feeding conditions or 16 weeks under simulated storage conditions. Concentrations of 2-AAF in the food were verified at each dose level before the food was fed to the animals. The allowable deviation from target dose was 10%. Batches found outside specifications were not used. Animals. Both sexes of the inbred mouse strain, BALB/cStCrllC3Hf/ Nctr (BALB/c), were used in the study. Mice were obtained from the breeding colony of the National Center for Toxicological Research as weanlings. Experimentul design. The basic experimental design as described previously (Fullerton et aZ., 199 1) was used, with modifications. The design was repeated with the two diets (AIN-76A and NIH-07). 2-AAF was administered in both diets at concentrations of 0, 100, 125, or 150 ppm to females and 0,20,40, or 60 ppm to males. The 2-AAF concentrations were selected for each sex to produce approximately 20, 50, and 80% bladder tumors at 104 weeks when administered with the nonpurified diet (Haley et al., 1974a,b; Littlefield et al., 1980; Farmer et al., 1980). Each dose group was composed of 96 weanling mice. At no time did feed mixed with 2-AAF remain in storage for more than 8 weeks nor did it remain in use in the feeders more than 7 days. All treatment groups were terminated after a 2-year (728-732 days) exposure to test diet with the exception of the highest dose groups of both sexes.When it became apparent that the AIN-fed mice receiving the highest 2-AAF concentration would not survive a full 2 years, both AIN- and NIHfed groups were scheduled to be euthanized at a time when 10 to 15% of

the AIN-fed mice would still be alive. Thus, the groups of male mice fed 60 ppm 2-AAF in either AIN or NIH diets were euthanized after 82 weeks of exposure; female groups fed 150 ppm 2-AAF were euthanized after a 92week exposure. Mice were killed by carbon dioxide anesthesia after a 16-hr fast and processed as described previously (Fullerton et al., 199 1). Statistical methods. The SAS procedure CHRONIC (Kodell et al., 1983) was used to analyze the liver and urinary bladder neoplasia data. Of primary interest was the comparison of the two diets, with respect to these neoplastic responses, to determine the influence of diet on both background and 2-AAF-induced neoplasms. Statistical comparisons between diets were made within each sex and each concentration of 2-AAF. Statistically, the presence of liver or bladder neoplasm was not considered the cause of death in any case. The procedure CHRONIC produces an age-adjusted prevalence function which is tested against the heterogeneity alternative (two-tailed test for a difference between diets) and also for positive trend (one-tailed test for a higher tumor incidence among animals fed the AIN-76A diet). A repeated measures analysis of variance technique was used to compare body weight gains and food consumption between test groups within each sex and between common dose levels within each sex.

RESULTS

Growth and Food Consumption Body weight gain of males and females was clearly influenced by diet. This is illustrated in Table 1. Statistical comparisons between the two diets at 4, 16, 32, and 64 weeks showed mice fed the AIN diet gained weight more rapidly

TUMORS

IN BALB/c

MICE

FED

for each 2-AAF concentration than did the NIH-fed mice (p < 0.01). Also, both male and female mice fed the AIN diet reached higher maximum body weights than their counterparts fed the NIH diet (p < 0.001, data not shown). Weight gain was not influenced by 2-AAF in female mice fed the NIH diet. However, at 32 and 64 weeks, body weight gain of females fed the AIN diet was clearly depressed by 2-AAF (p < 0.01) at the two highest dose levels (125 and 150 ppm). It is noteworthy that inhibition of weight gain by 2-AAF at 150 ppm in females receiving the AIN diet resulted in body weight gain curves that were very similar to those of females receiving the same concentration of 2-AAF admixed with the NIH diet. Male mice fed the NIH diet showed a linear 2-AAF dose trend for reduced weight gain at 16 and 32 weeks (p < 0.05). At the levels tested, 2-AAF had no effect on weight gain of males fed the AIN diet. Food consumption data, representative of selected time periods during the study, are summarized briefly in Table 2. There were no 2-AAF dose effects on food consumption with either sex. During the early part of the study, particularly the first week, males and females fed the AIN diet consumed more food than those fed the NIH diet (p < 0.0001). Subsequently, both sexes fed the NIH diet ate more than those fed the AIN diet (p < 0.002) until approximately 44 weeks. From that point until the end of the study AIN-fed males ate more than their NIH-fed counterparts (a < 0.0001). Also, starting at about 68 weeks (data not shown), females fed the AIN diet ate more than those fed the NIH diet (p < 0.000 1). Mortality

Table 1 shows the median lifespan of BALB/c males and females for each diet and 2-AAF dose level. The median lifespan of each NIH-fed group was longer than for the corresponding sex and 2-AAF dose group fed the AIN diet (p < 0.001). None of the NIH-fed groups of males reached 50% mortality before being terminated at 730 days while all of the AIN-fed male groups reached 50% mortality before 565 days. Fifty percent of the high 2-AAF-dosed AIN-fed males died by 448 days, whereas only 9% of their NIH-fed counterparts had died. It was necessary to euthanize both diet groups fed 60 ppm 2-AAF at 575 days in order to obtain a diet comparison of tumor prevalence at a single point in time. All other dose groups of males were euthanized at 24 months. There was little or no effect of 2-AAF on median lifespan at the two lower doses (20 or 40 ppm) in BALB/c males within either diet. All groups of BALB/c females fed either diet reached 50% mortality prior to termination at 730 days. Diet had considerably less effect on mortality in females than in males. Mortality among BALB/c females fed the NIH diet showed a slight trend for a 2-AAF dose response (p = < 0.05) while mortality between 2-AAF dose groups fed AIN diet was definitely dose-related (p < 0.001). Both diet groups of females fed 150 ppm 2-AAF were terminated at about 22.5 months

AIN-76A

OR

NIH-07

195

DIET

because of high mortality in the AIN-fed group. All other dose groups were euthanized at 24 months. Liver Tumors A summary of the incidence of liver neoplasia is presented in Table 3. Benign neoplasms were hepatocellular adenomas or hemangiomas. Malignant liver neoplasms were predominantly hepatocellular carcinomas; however, one cholangiocarcinoma, two hepatoblastomas, and one hepatocholangiocarcinoma were also found. One hepatoblastoma was found in a control male fed AIN diet, the other in an AIN-fed female receiving I25 ppm 2-AAF. Other malignancies were occasionally found in the liver, including malignant lymphomas and metastatic tumors from other sites, as well as malignant tumors of blood vessels (hemangiosarcoma). Those liver neoplasms that were not primary were excluded from analysis. Diet had a significant impact on the frequency of liver neoplasia (Table 3). Controls of both sexes fed the AIN diet had a higher frequency of liver neoplasms than NIH-fed controls. This was also true of malignant liver neoplasms in males (p = 0.035) but not in females (p = 0.126). There was no apparent effect of diet on the proportion of malignant to benign liver neoplasms in either sex. 2-AAF did not increase the frequency or malignancy of liver neoplasms in males fed either diet (Table 3). However, liver neoplasms were more frequent in every male treatment group fed the AIN diet than in their NIH-fed counterparts. On the other hand, 2-AAF clearly increased the frequency of liver neoplasia in females fed either diet, and AIN-fed females responded to 2-AAF more quickly and at higher frequency than NIH-fed females. While this diet effect was noted at all 2-AAF dose levels it was most apparent at the lowest dosage.

Bladder and Kidney Tumors

Urinary bladder tumors were induced by 2-AAF in both sexes but diet influenced this process only in males (Table 4). Clearly males fed either diet were more sensitive to bladder tumor induction by 2-AAF than females. Furthermore, male mice fed the AIN diet were more sensitive to bladder tumor induction by 2-AAF (at levels of 40 ppm and above) than their NIH-fed counterparts by a highly significant margin (p < 0.0001). Neoplasms were infrequent in the kidney and did not appear to be diet related. DISCUSSION

AND CONCLUSIONS

A comparison of BALB/c control mice fed the purified diet with those fed the nonpurified diet revealed several significant differences. The AIN diet was associated with increased weight gain, shortened lifespan, and increased “spontaneous” liver tumor incidence in both sexes. We have reported similar findings in B6C3Fl mice (Fullerton et al., 1991).

196

FULLERTON,

GREENMAN.

AND BUCCI

TABLE 2 Weekly Food Consumption of BALB/c Mice Fed AIN-76A or NIH-07 Diet Weekly food consumption (g/g body weight) 2-AAF concn. Weeks on study:

Sex

Diet

F

AIN

0 100 125 150

1.38 + 0.13 1.41 f 0.18 1.44 + 0.16 1.39 _+0.16*

0.88 0.92 0.93 0.90

+ i rt k

0.07 0.09 0.06 0.08’

0.81 0.83 0.80 0.81

f + + *

0.10 0.11 0.07 0.07’

0.72 0.81 0.82 0.83

+ + f +

0.05 0.12 0.09 0.08

F

NIH

0 100 125 150

1.20 1.15 1.12 1.13

0.92 0.95 0.94 0.98

+ f f +

0.12 0.13 0.07 0.09

0.85 0.87 0.84 0.85

f 2 + +

0.07 0.09 0.07 0.08

0.81 0.81 0.78 0.85

+ + f f

0.09 0.13 0.10 0.10

M

AIN

0 20 40 60

1.25 + 0.12 1.29 + 0.14 1.32 + 0.16 1.29 f 0.14b

0.71 0.74 0.70 0.70

f 0.08 zk 0.06 * 0.05 + o.046

0.70 0.70 0.69 0.69

f 0.08 IL 0.07 + 0.08 f 0.07’

0.82 0.83 0.87 0.94

* + f +

0.11 0.09 0.16 0.156

M

NIH

0 20 40 60

1.05 1.04 1.03 1.06

0.85 0.87 0.86 0.86

+ f f f

0.81 0.77 0.77 0.81

k 0.14 k 0.11 f 0.07 ~fr0.06

0.74 0.73 0.78 0.75

f 0.08 z!z0.07 f 0.15 f 0.08

(mm)

I

f + + f

+ * rt +

16

0.24 0.12 0.08 0.13

0.15 0.08 0.08 0.10

0.11 0.12 0.12 0.11

32

64

’ The data are presented as the mean + SD. b Univariate analyses between diets at this time period indicate significantly different from NIH-fed mice of corresponding sex (p < 0.0001). ’ Univariate analyses between diets at this time period indicate significantly different from NIH-fed mice of corresponding sex (p < 0.002).

In addition to effects in control BALB/c mice, the AIN diet tended to enhance the toxicity of 2-AAF. For example, 2-AAF inhibited weight gain, shortened lifespan, and at the highest dose enhanced liver carcinogenesis more in females fed the AIN diet than in females fed the equivalent dietary concentrations of 2-AAF with the NIH diet. An increase in 2-AAF toxicity was demonstrated in males fed the AIN diet by a shortened lifespan and increased sensitivity to bladder tumor induction. At approximately 18 months into the study it was learned that several batches of the AIN diet were deficient in thiamine (approximately one-tenth of the specified level). The animals received the thiamine-deficient diet about 8 weeks. All batches of the diets were analyzed during the study and all except those fed during this 8 week period contained thiamine within the recommended levels (NRC, 1978). That thiamine deficiency played some role in the increased toxicity of 2-AAF cannot be ruled out (since body weight loss occurred during this period). However, mortality curves (not shown here) clearly show that the death rate had started to increase among AIN-fed mice long before the thiamine deficiency occurred. Thus, early deaths were not related to thiamine deficiency while body weight loss clearly was. Since thiamine deficiency occurred during a relatively brief period very late in the tumorigenic process no effect on the initiation of tumorigenesis would be anticipated. In addition, a search of the literature has failed to reveal evidence that thiamine deficiency exerts an influence on the tumorigenic process

although a deficiency great enough to cause body weight loss might be expected to slow tumor progression. Thus, it seems probable that AIN-related increases in tumorigenicity were a true effect of the diet and occurred in spite of rather than because of thiamine deficiency. Alterations in 2-AAF toxicity by the AIN diet may be partially explained as a consequence of changes in the metabolism of 2-AAF. Although we have not attempted to demonstrate a diet-induced change in 2-AAF metabolism, we have reported differences in the metabolism of estradiol by mice fed these two diets (Fullerton et al., 1987) that could explain an increase in the response of estrogen target tissues to estradiol administration when animals were fed the AIN diet (Greenman and Fullerton, 1986). Since numerous enzymes are involved in 2-AAF metabolism (Maleijka-Giganti et al., 1989; Astrom and DePierre, 1985), multiple sites of dietary influence are possible, and the balance of these effects must, in general, enhance 2-AAF toxicity on carcinogenicity in response to the AIN diet. If AIN-enhanced sensitivity of males to bladder tumor induction could be explained by changes in metabolism of 2-AAF, the fact that this enhancement does not occur in females suggests that the AIN diet probably causes quite different effects on 2-AAF metabolism in the two sexes. In rats it is clear that there are sex differences in the metabolism of 2-AAF (Maleijka-Giganti et al., 1989). Further, treatment of male and female rats with P-naphthoflavone has been shown to have opposite effects on the production of one

TUMORS

Liver Neoplasia

197

IN BALB/c MICE FED AIN-76A OR NIH-07 DIET

TABLE 3 in BALB/c Mice Fed AIN or NIH Diet Containing Graded Levels of 2-AAF Frequency”

Sex

2-AAF in diet @pm)

F

0

F

100

F

125

F

150

M

0

M

20

M

40

M

60

Euthanized b

Dead or moribund Diet

Primary’

Malignantd

N’

PrimaryC

Malignantd

N’

Significance

w

NIH AIN NIH AIN NIH AIN NIH AIN NIH AIN NlH AIN NIH AIN NIH AIN

0 1.7 25.0 48.7 28.2 38.4 27.7 26.0 7.1 19.2 2.7 16.5 7.9 13.9 0 21.9

0 1.7 15.6 26.9 14.1 19.8 19.1 13.7 7.1 9.6 2.7 3.8 2.6 6.3 0 5.5

43 59 64 78 71 86 47 73 28 73 37 79 38 79 8 73

2.0 12.1 40.0 100.0 60.0 80.0 31.1 77.8 4.6 26.3 5.3 45.5 12.5 44.4 1.1 21.4

0 0 20.0 76.9 36.0 80.0 11.1 33.3 1.5 10.5 0 18.2 1.8 22.2 0 7.1

108 33 30 13 25 7 45 9 65 19 57 11 56 9 87 14

0.01709g

(0.034 1)

0.00005

(0.000 1)

0.00025

(0.0005)

0.022 I I

(0.044 1)

0.00083 h

(0.0016)”

0.00009

(0.0002)

0.00223

(0.0044)

0.00005

(0.000 I)

’ Expressed as percentage with Neoplasm. b Most groups were euthanized after 2 years on study. The only exceptions were the groups of females dosed with 150 ppm 2-AAF, euthanized at 647 days and the groups of males dosed at 60 ppm 2-AAF, euthanized at 574 days. ’ Percentage of mice with primary liver neoplasia (nonmalignant + malignant). Nonmalignant neoplasms were primarily hepatocellular adenomas but also included hemangiomas. d Percentage of mice with malignant liver neoplasms. Malignant neoplasms were predominately hepatocellular carcinoma. One cholangiocarcinoma was found in a NIH-fed female receiving 100 ppm 2-AAF. One hepatoblastoma was found in a control AIN-fed male and one in an AIN-fed female treated with 125 ppm 2-AAF. One hepatocholangiocarcinoma was found in an AIN-fed female receiving 125 ppm 2-AAF. ‘N, number of mice examined. Ip-values are for comparisons of age-adjusted prevalences of total liver neoplasms. Except where noted. pvalues were similar where malignant neoplasms alone were tested. Nonparenthetic p-values are for one-tailed comparisons hypothesizing that the tumor incidence was greater when the AIN diet was fed; pvalues in parentheses are for two-tailed tests for any dietary difference. g pvalue for malignant neoplasms was 0.126. ‘p-value for malignant neoplasms was 0.035.

metabolite (9-hydroxy-2-AAF) of 2-AAF in the two sexes (Maleijka-Giganti et al., 1989). While altered 2-AAF metabolism may have caused the enhancement of induced liver tumors in females, it should be noted that high dietary sucrose reportedly promotes the development of enzyme-altered liver foci following initiation by a chemical carcinogen (Hei and Sudilovsky, 1985). Thus, the AIN diet, which contains 50% sucrose, might act as a liver tumor promoter when compared to the NIH diet without necessarily causing a change in 2-AAF metabolism. Further, whatever its mode of action, the AIN diet clearly enhances the development of “spontaneous” liver tumors in both sexes, suggesting that its effect on liver tumors induced by 2-AAF in females may not be related to a change in 2-AAF metabolism. The fact that the two higher doses of 2-AAF were associated with lower liver tumor incidence than that found at 100 ppm in AIN-fed female mice may be due partially to early mortality in AIN-fed groups receiving higher 2-AAF

concentrations, thus reducing the time for development of tumors. Furthermore, the fact that the 150-ppm-treated group was killed earlier than other groups due to high death rate undoubtedly reduced the tumor incidence in this group. Thus, animals treated with lower 2-AAF levels had more time to develop tumors. In addition, at the two higher doses weight gain was clearly inhibited. The lower weight gain at these higher doses may have been causally related to suppression of overall liver tumor incidences. This is especially suggested by the observation that liver tumor incidence and body weights were very nearly the same in AINfed and NIH-fed females receiving 150 ppm 2-AAF. In comparing results of the current study with those previously reported for B6C3Fl mice, it can be generally stated that compared to the NIH diet, the AIN diet shortened the lifespan, enhanced body weight gain, and tended to increase liver and urinary bladder neoplasia in both BALB/c and B6C3Fl mice (Fullerton et al., 1991). Nevertheless, there were distinct strain differences in response to the two diets.

198

FULLERTON,

GREENMAN, TABLE

AND BUCCI

4

Bladder Neoplasia in BALB/c Mice Fed AIN or NIH Diet Containing Graded Levels of 2-AAF Frequency LI

Sex

2-AAF in diet (mm)

F

0

F

100

F

125

F

150

M

0

M

20

M

40

M

60

Dead or moribund

Euthanizedb

Diet

Primary’

Malignant d

N’

Primary’

NIH AIN NIH AIN NIH AIN NIH AIN NIH AIN NIH AIN NIH AIN NIH AIN

2.4 1.9 27.9 29.9 72.9 63.5 78.7 78.1 0 0 0 4.1 22.9 53.2 12.5 89.5

2.4 0 9.8 15.6 48.6 44.7 61.7 63.0 0 0 0 1.3 11.4 14.3 12.5 69.7

42 54 61 77 70 85 47 73 27 69 34 75 35 77 8 76

0 0

0 0

40.0 46.2 92.0 80.0 88.6 66.7 0 0 0 9.1 26.8 100.0 64.4 100.0

32.3 15.4 48.0 40.0 45.5 22.2 0 0 0 0 8.9 22.2 25.3 42.9

Malignantd

NC

Significance

(P)’

50 33 30 13 25 5 44 9 63 19 57 11 56 9 87 14

0.40141

(0.8028)

0.10914

(0.2181)

0.53572

(0.9286)

0.38672

(0.7734)

0.04772

(0.0953)

0.01520

(0.0303)

0.00005

(0.0001)

0.00005

(0.000 I)

’ Expressed as percentage with neoplasm. ’ Most groups were euthanized after 2 years on study. Only exceptions were the groups of females dosed with 150 ppm 2-AAF, euthanized at 647 days, and the groups of males dosed at 60 ppm 2-AAF, euthanized at 574 days. ’ Percentage of mice with primary bladder neoplasia (nonmalignant + malignant). Nonmalignant neoplasms were papillomas. d Percentage of mice with malignant bladder neoplasms. Malignant neoplasms included transitional cell squamous cell carcinomas. ’ N, number of mice examined. /Nonparenthetic p-values are for one-tailed comparisons hypothesizing that age-adjusted total tumor incidence was greater when the AIN diet was used; p-values in parentheses are for two-tailed tests for any dietary difference.

For example, the AIN diet had less life shortening effect in BALB/c mice than in B6C3Fl mice. Further, stimulation of body weight gain by the AIN diet was much less in BALB/c than in B6C3Fl mice. Also, there was a much more dramatic effect of the AIN diet on spontaneous liver neoplasia in B6C3Fl females, and the AIN diet clearly enhanced 2-AAF-induced bladder tumorigenesis in B6C3Fl females but had no effect on bladder tumorigenesis in female BALB/c mice. In contrast, the AIN diet enhanced bladder tumorigenesis and spontaneous liver neoplasia in BALB/c males more than in B6C3Fl males. These data indicate the importance of diet in toxicological and carcinogenic studies. They also point out differences in strain response to diets and treatment. Although the use of purified diets generally increases the sensitivity of animals to chemically induced carcinogenesis they also increase spontaneous carcinogenesis and may increase other types of toxicity. Thus, while their use is essential in studying the effect of nutrition on the carcinogenic process it is important to recognize complications introduced be the use of such diets. ACKNOWLEDGMENTS The authors gratefully recognize the assistance of Ms. Ruth York in preparation of the manuscript. Mr. Charles McCarty and Mr. Scott Jordan are

recognized for their assistance in statistical analysis of the data. Animal care was ably provided by Ms. Gina Wood, Ms. Liz Conner, Mr. Bob Harmon, and Ms. Kristina Stephens. REFERENCES Ad Hoc Committee on Standards (1977). Report of the ad hoc committee on standards for nutritional studies. J. Nutr. 107, 1340- 1348. Astrom, A., and DePierre, J. W. (1985). Metabolism of2-acetylaminofluorene by eight different forms of cytochrome P-450 isolated from rat liver. Carcinogenesis 6, 113- 120. Campbell, T. C., and Hayes, J. R. (1974). Role of nutrition in the drug metabolizing enzyme system. Pharmacol. Rev. 26, 17 l- 197. Carroll. K. K., and Khor, H. T. (1975). Dietary fat in relation to tumorigenesis. Prog. Biochem. Pharmacol. 10, 308-353. Doll, R. (1979). Nutrition and cancer: A review. Nufr. Cancer l(3), 45-75. Farmer, J. H., Kodell, R. L., Greenman, D., and Shaw, G. W. (1980). Dose and time response models for the incidence of bladder and liver neoplasms in mice fed 2-acetylaminofluorene continuously. .I. Environ. Pafhol. Toxicol. 3, 55-68.

Fullerton, F. R., Greenman, D. L., McCarty, C. C., and Bucci. T. J. (1991). Increased incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in B6C3Fl mice fed AIN-76A diet versus NIH-07 diet. Fundam. Appl. Toxicol. 16, 5 l-60. Fullerton, F. R., Greenman, D. L., and Young, J. F. (1987). Influence of a purified diet and route of administration on the metabolism and disposition of estradiol in B6C3Fl mice. Drug Metab. Dispos. 15, 602-607. Greenman, D. L., and Fullerton, F. R. (1986). Comparison of histological responses of BALB/c and B6C3Fl female mice to estradiol when fed

TUMORS

IN BALB/c MICE FED AIN-76A OR NIH-07 DIET

purified or natural-ingredient diets. J. Toxico[. Environ. He&h 19, 53!540. Greenman, D. L., Oiler, W. L., Littlefield, N. A., and Nelson, C. J. (1980). Commercial laboratory animal diets: Toxicant and nutrient variability. J. Toxicol.

Environ.

Health.

6, 235-246.

Haley, T. J., Schieferstein, G., Harmon, J. R., Dooley, K. L., Jaques, W. E., Frith. C., and Farmer, J. H. (1974a). Ninety day subchronic toxicity of N-2-fluorenylacetamide (FAA) in C57BL/6J and BALB/cStCrIBR mice (38164). Proc. Sot. Exp. Biol. Med. 146,648-65 1. Haley, T. J., Schieferstein, G., Jaques, W. E., Farmer, J. H., Frith, C., and Sprawls, R. W. (1974b). Dose response predictability of urinary bladder hyperplasia by N-2-fluorenylacetamide feeding in mice: Its modification by sex. J. Pharm. Sci. 63, 1946-1947. Hei. T. K., and Sukilovsky (1985). Effects of a high sucrose diet on the development of enzyme-altered foci in chemical hepatocarcinogenesis in rats. Cancer Res. 45, 2700-2705. Institute of Laboratory Animal Resources Committee on Revision of the Guide for Laboratory Animal Facilities and Care (1972, revised). Guide for the Care and Use qfLaboratory Animals, DHEW Publication (NIH) 74-23. U.S. Department of Health, Education, and Welfare, Washington, D.C. Institute of Laboratory Animal Resources Committee on Laboratory Animal Diets (1978). Control of diets in laboratory animal experimentation. ILAR News 21, Al-A12.

199

Kode!!, R. L., Haskin, M. G., Shaw, G. W., and Gaylor, D. W. (1983). CHRONIC: A SAS procedure for statistical analysis of carcinogenesis studies. J. Statist. Comput. Simul. 16, 287-3 10. Kritchevsky, D. (1986). Diet, nutrition, and cancer-The role of fiber. Cancer 58, 1830-1836. Littlefield, N. A., Farmer, J. H., Gaylor, D. W., and Sheldon, W. G. (1980). Effects of dose and time in a long-term, low-dose carcinogenic study. J. Environ.

Pathol.

Toxicol.

3, 17-34.

Maleijka-Giganti, D., Magat, W. J., and Decker, R. W. (1989). Sex hormonemediated effectson the phase I and phase II metabolism of N-2-fluorenylacetamide: Modulation of 9-hydroxylation. Biachem. Pharmacol. 38, 1075-1082. National Cancer Institute. (198 I). Workshop on fat and cancer, Bethesda, MD, December 10-12, 1979. Cancer Res. 41(9, Part 2). National Research Council (1978). Nutrient Requirements of Laboratory Animals, No. 10, 3rd revised ed. National Academy of Sciences, Washington, D.C. Newbeme, P. M., and Conner, M. W. (1986). Nutrient influences on toxicity and carcinogenicity. Fed. Proc. 45, !49- 154. Topham, J. C., and Eva, J. K. ( 198 1). A quality controlled rodent diet. Lab. Anim. l&97-100. Wiseburger, J. H. (1979). Mechanism of action of diet as a carcinogen. Nutr. Cancer

l(2),

74-82.

Wise, A. (198 1). The standard stock diet-Fact 23,287-293.

or fiction. Nutr. Rep. Inst.