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A Comparison of the Lung Adenoma Response in Strain A/J Mice after lntraperitoneal and Oral Administration of Carcinogens

GARY D. STONER.* ELIZABETH A. GREISIGER,* HERMAN A. J. SCHUT,* MICHAEL A. PEREIRA,~ TIMOTHY R. LOEB,* JAMES E. KLAUNIG,*

AND DANIEL G. BRANSTETTER*

*Depart,nent c?f Pathology, Medical College q/Ohro. 3000 ~r i ing ton Avenue. Toledo, Ohro 43614, and tl?.S. Envrronmental Protection Agency, Health Effects Research Laboratory.

26 U'est St. Clair Street. Cincinnatr, Ohro 45268

Rece~ved Ma), 14. 1983: accepted August 31. 1983

A Comparison of the Lung Adenoma Response in Strain A/J Mice after Intraperitoneal and Oral Administration of Carcinogens. STONER. G. D., GREISIGER, E. A,, SCHUT, H. A. J.. PEREIRA. M. A., LOEB, T. R.. KLAUNIG, J . E., A N D B R A X S T E ~ E R . D. G. (1984). Trnrcol. .4ppl Pharmac-ol 72, 3 13-323. This study was undertaken to compare the abil~ty of a series of compounds from different chemical classes to induce lung tumors In strain A/J mice after either ip or po administration. 3-Methylcholanthrene, benzo(a)pyrene, urethan, diethylnitrosam~ne. ethylnltrosourea. and dimethylhydrazine induced a significant ( p < 0.05; t test) increase in the lung tumor response when given both ip and po. 2,4-Dinitrotoluene. 2,6-dinitrotoluene. and a ? : I mixture of 2.4- dinitrotoluene and 2.6-dinitrotoluene were inactive by both routes of administration and at all dose levels. The lung tumor response to all doses of 3-methylcholanthrene and benzo(a)pyrene, the highest dose of diethylnitrosamine. and the middle doses of both ethylnitrosourea and dimethylhydrazine varied as a function of the route of administration. This finding was most evident for the polycyclic hydrocarbons, e.g.. the average number of lung tumors per mouse in animals that received the middle dose of 3-methylcholanthrene or the highest dose of benzo(a)pyrene by the ip route exceeded that by the po route by factors of 12 and 13, respectively. Tissue distribution and elimination studies were conducted in an effort to determine the basis for the observed difference in lung tumor response to 3-methylcholanthrene after ip or po administration. The data indicated that 3-methylcholanthrene persists for longer periods in the animals when given ip, thus potentially providing an extended carcinogenic st~mulus. Extra- pulmonary lesions observed at a higher than normal frequency at necropsy included peritoneal sarcomas (in 3-methylcholanthrene-treated mice), and both squamous cell carcinomas of the forestomach and abnormal lesions of the liver (in diethylnitrosamine-treated mice).

The strain A mouse lung tumor bioassay for testing the potential carcinogenic effects of en- vironmental chemicals was developed in 1940 (Shimkin, 1940: Andervont and Shimkin, 1940). Subsequently, more than 200 compounds have been tested for carcinogenic activity in the bioassay (Shimkin and Stoner, 1975; Stoner and Shimkin, 1982). Most studies have employed the ip route for administration of the test chemicals to the mice (Shimkin and Stoner, 1975; Stoner and Shimkin, 1982).

However, lung tumors have been induced in strain A mice when carcinogens were given sc (Shimkin, 1940; Heston, 1940), iv (Shimkin. 1940; Andervont and Shimkin, 1940). by inhalation (Leong, el al.. 1971). and when painted on the skin (Murphy and Sturm, 1925).

With the exception of an early study in which polycyclic hydrocarbons were found to be more carcinogenic after iv administration than when given sc (Shimkin, 1940), there has

004 1 -008x184 $3.00 Copbnghr C 1984 b\ Arademlc Press in' All nghls of reprcducuon In an, form resened

STONER ET AL.

been no systematic cornparison of the lung With sterile technique. the chemicals were administered

response in strain A to carcinogens either ip by injection or po (gavage) as 0.1 mlldose. in walq or in tecapryljn (glycerol trioctanoate, Eastman

given by various routes' i n the Kodak. Rochester, N.Y.). Solutions were freshly prepared present study, a comparison was made of the immediately before administration. Amber colored bottles carcinogenic potential of a series of corn- were used to protect the chemicals from fluorescent I~ght.

pounds from different chemical classes (poly- Preliminarj. loxicology. Maximum tolerated doses

cyclic hydrocarbon, nitrosarnine, nitrosourea, (MTD) for BP. ENU, DMH, 2,4-DNT3 2,6-DNT9 and the 2:l mixture of 2.4-DNT and 2,6-DNT were deter-

carbarnate, hydrazine3 and nitrotoluene) to mined. Serial twofold dilutions of each chemical or the induce lung tumors in strain A/J mice when mixture were administered either ip or po to groups of given by at least two routes: ip and po. An eight mice (four males and four females). The MTD was

ip-po cornparison seemed appropriate since the maximum dose that all eight mice tolerated (survived)

most long-tem, chronic bioassays have ern- after receiving 3 ip or po treatments over a I-week period. Animals were held for 1 month before experimental groups

the route and data were initiated in order to detect delayed toxicity. between the lung and long- After ip administration, MCA and urethan have been term bioassays are desirable. shown to induce lung tumors in strain A mice (Shimkin

Results from the present study indicated el al., 1969; Stoner el a/.. 1974), and DENA induced lung

that the polycyclic hydrocarbons, 3-methyl- tumors in adult SWR mice (Minish and Kaufman. 1970). ---------- Therefore. in the present study, the doses used for MCA. ''' '.' - r e n d s A ) and & n z o l a j u e n ! urethan, and DENA were selected from the published @yL as well as other cornpou<ds were sig- data,

p \>'I? ? 3 nificantly --.. - ~ . . more carcinoge.njc when given, lp B~oas.ra)s. Each compound was tested at three dose @an-.~po. Therefore, for MCA. tissue di&-' levels: the MTD. 0.5 MTD, and 0.2 MTD. with either

butioc-gnd studies were con- approximately 32 or 52 mice (equal number of males and females) per dose. MCA. BP, urethan. DMH. and ENU

ducted to determine the basis for the observed were given in a single injection; was administered difference in lung tumor response. once weekly for 4 weeks; and the dinitrotoluenes were

given either three times per week for 8 weeks (ip) or two times per week for 12 weeks (po). Control groups consisted

METHODS of untreated and vehicle (water or tricaprylinttreated mice. Animal weights were obtained every 2 weeks during the

An~mals. Six- to eight-week-old male and female mice injection period and at monthly intervals thereafter. were used in the bioassays. They were obtained from our Mice given MCA. BP. DENA, urethan. DMH, or ENU breeding colony, derived from A/J mice purchased from were lulled 24 weeks after initiation of the bioassay. An- the Jackson Laboratories. Bar Harbor. Maine. The mice imals that received either 2.4-DNT, 2.6-DNT. or the 2:l were kept on corn cob bedding in temperature ( 2 2 ' 0 mixture of 2.4-DNT and 2,6-DNT were killed after 30 and humidity (50%) controlled rooms with a 12-hr light/ weeks. The lungs were removed and fixed for 24 hr in dark cycle. NIH-07 certified rodent diet (Ziegler Brothers. 70% ethanol containing 5 4 glacial acetic acid and 5% Gardner. Penn.) and water were provided ad librrum. The formaldehyde. Lung tumors. which appeared as p e ~ k - ~ health status of the breeding colony and the mice used w>te-nodules the surface,-of \helun_g (Shimkin and in the bioassays was periodically examined by (a) complete Stoner, 1975). were counted and randomly sampled for -. gross necropsy. tabulating gross lesions. and general con- hixt-?pathol.opcal evaluat~on a n ~ 6 ~ ~ f i a ~ o n o f a d e n o m a . .. . _ _ ^ _ . _ I -..-. - - . dition and body weight of each animal: (b) histopatho- Duplicate lung tumor counts were obtarneaby two tech- logical evaluation of formalin fixed tissues involving all nicians working independently. In addition. the liver. kid- major organs: and (c) serologcal tests for the presence of neys, spleen, intest~nes. thymus. stomach, and thesalivary murine virus infections. and endocrine glands were examined grossly. If gross le-

Chemicals. Reagent grade MCA, BP. diethylnitrosamine sions were observed, they were examined histologically (DENA), urethan. d~methylhydrazine (DMH), and ethyl- for the presence of neoplasms. nitrosourea (ENU) were purchased from Sigma Chemical The lung tumor response (percentage of mice that de- Co.. St. LOUIS. Mo. The nitrotoluene compounds. 2.4- veloped lung tumors and the number of lung tumors per d~nitrotoluene (2.4-DNT: 92-95% pure with the major mouse) in experimental and control groups was compared impunty being 2,6-dinitrotoluene) and 2.6-dinitrotoluene by Student's I test. (2.6-DNT: 98% pure), were supplied by Dr. Mary C. T~ssur disrriburion and eliminar~on s~udies w,irh MC4. Henn. U.S. Army Medical Bioengneering Research and [G-'HIMcA (SA 26 Ci/mmol) was obtained from Amer- Development Laboratory, Fort Detrick, Frederick, Md. sham (Arlington Helghts, 111.) and was determined to be The chemicals were stored at 4°C in the dark. >98.2% radiochemically pure as judged by high-pressure

. Eastman y prepared red bottles scent light lted doses DNT. and vere deter- ical or the groups of MTD was ( s u ~ i v e d )

eek period. ntal groups ity. have been

z (Shimkin duced lung nan. 1970). 1 for MCA.

published

three dose with either f males and . and ENU lministered uenes were (ip) or two x consisted zated mice. during the

reafter. H. or ENU sassaq. An- , or the 2: 1 ed after 30 )r 24 hr in :id and 5% I as pearly- ~ imkin and ampled for )f adenoma. y two tech- le liver. kid- the salivar) If gross le-

stologically

ice that de- tumors per s compared

rom Amer- nined to be gh-pressure

LUNG ADENOMA BIOASSAY IN A/J MICE 3 15

liquid chromatography (HPLC) (Ultrasphere ODS col- cedures. The corrected dpm of I'HIMCA was then ex.

101. [6-I4C]MCA(SA 58.5 mCi/mmol) was purchased from New England Nuclear (Boston. Mass.) and was de- I

thetized with diethyl ether. Their blood. urine, and tissues of lung adenomas in untreated and vehicle- 1

upon opening the peritoneum, all organs were carefully blotted to remove any unabsorbed material. ferent ( p > 0.05, t test); rnererore, resulrs rrom

In additional studies, the administered MCA was re- lhe two isolated. Mice were injected with ['HIMCA as described treated mice represent the "s~ontaneous" oc- above. The liver, lungs, and small intestine were removed. currence of lung tumors in A/J mice and agree welghed, and then homogenized in 5 volumes of ice-cold with earlier results on of equivalent age phosphate-buffered saline. Immediately before extraction. (shimkin 1960. c+,,-,.- -, - 1 I 0 7 2 .

MCA (200 pg/sample) and ['4C]MCA ( 10.000 dpm/sam- ,. . . . . - .

, , r .

graphed on thin-layer silica gel G F plates (Fisher Scientific, i n *he untreated micr ;-A;--+; 250 pm, with fluorescent indicator). which were developed with benzene:n-hexane (1:I). Under these conditions. currence of lung MCA migrated with an R,of 0.8 and was well separated the injections of vehicle. from its metabolites, all of which had lower R, values. Data on bioassays of the eight chemicals The MCA area was marked under uv light and scraped following ip or po arlrninirtratinn rrrp clrrn- into scintillation vials. After adding 20 ml of organic scin- marized in Table 2 . tillation mixture, the vials were counted bv standard dou-

jncurred during the extraction and chromatography pro- therefore, data from the two sexes were com-

TABLE 1

LUNG TUMOR RESPONSE IN UNTREATED AND VEHICLE-TREATED A/J MICE

Number of

Treatment Route treatments

Duration of experiment

(weeks) Su~vivors/

initial

lung tumors

(70)

Untreated - - Untreated - - Tricaprylin ip 1 Tricaprylin ip 24 Tricaprylin PO I Tricaprylin PO 24 Water ip I

" Combined data from males and females. Values are mean ? standard error.

number lung tumors/ mouse

0.35 * 0.06' 0.38 ? 0.10 0.28 5 0.08 0.37 & 0.09 0.28 5 0.06 0.31 t 0.08 0.28 k 0.07

STONER ET AL.

. . . . . C i b e e C L C C P / i / * k c

F"s ;s L & g &,= r u m - onm m m m & L b & & $ hl oqy- Y ? = 9 1 0 ? ~ r n ?

m n o - o z 3 d d d d l c r o o o o o - 0 0 - 0 0 - - o

ENU (ip)

ENU (po) T 24 100 32 32 32 100 17 t 1.93' 60.7 50 32 30 29 97 5 t 1.56' 17.9 20 32 3 1 27 87 2 t 0.21' 7.1

DMH (ip) W 24 2 5 3 1 3 1 13 42 0 . 7 1 t 0 . 1 8 ' 2.5 12.5 3 2 32 8 25 0.28 + 0.09 I .O 5 3 3 33 9 27 0.30 2 0.09 I. I

DMH (PO) w 24 2 5 32 16 7 44 0.75 t 0.25' 2.7 12.5 32 32 18 56 0.75 + 0.14" 2.7 5 32 32 16 50 0 . 5 3 t O . l O 1.9

2,4-DNT ( i ~ ) ~ T 30 3000 5 2 50 13 26 0.28 t 0.07 0.8 I500 52 5 2 9 19 0.2 I t 0.06 0.6 600 53 52 23 44 0.46 -t 0.08 1.2

2,4-DNT (po) T 30 6000 5 2 44 10 23 0.30 * 0.09 0.8 3000 5 2 48 15 31 0 . 3 1 t 0 . 0 7 0.8 1200 5 2 47 13 28 0.34 -t 0.08 0.9

2.6-DNT (ip) T 30 3000 5 2 47 14 30 0.40 t 0.10 1.3 1500 5 2 5 1 2 3 45 0.53 k 0.09 1.7 600 5 2 50 17 34 0.40 a 0.09 1.3

2.6-DNT (po) T 30 6000 5 2 38 13 34 0 . 4 7 a 0 . 1 2 1.5 3000 52 50 I I 22 0.26 3 0.07 0.8 1200 5 2 49 9 18 0.27 t 0.09 0.9

2,4-DNT:2,6-DNT (2: 1 ) T 30 4800 5 2 40 9 23 0.25 t 0.08 0.7 ( i ~ ) 2400 5 2 50 14 28 0.32 t 0.08 0.9

960 52 48 16 33 0.37 -t 0.08 1 .O

2.4-DNT:2,6-DNT (2: I) T 30 6000 5 2 48 16 33 0.40 0.09 1.3 (PO) 3000 52 48 17 35 0.40 k 0.08 1.3

I200 5 2 48 17 35 0.40 t 0.08 1.3

" Total cumulative dose per animal. MCA, 3-methylcholanthrene; BP, benzo(a)pyrene; DENA, diethylnitrosamine; ENU, ethylnitrosourea; DMH, dimethylhydrazine; DNT, dinitrotoluene. ' T, tricaprylin; W, distilled water.

MCA, BP, urethan, ENU, and DMH were given in a single administration; DENA was administered once weekly for 4 weeks; and the dinitrotoluenes were given either 3 times per week for 8 weeks (ip) or 2 times per week for I2 weeks (po).

'Significantly different (p < 0.05, 1 test) from corresponding vehicle treated control (Table I). 'Significantly different ( p < 0.05. I test) from corresponding po or ip group.

The ip data for 2,4-DNT were taken from Schut er a/. (1 982).

318 STONER ET AL.

bined. MCA, at a total dose of 100 mg/kg, treated groups are summarized in Table 3. was highly toxic when given ip but not po. There were no grossly appearing lesions other The animals were dying as late as 23 weeks than lung adenomas observed among the con- after receiving the single injection. All other trols or in mice administered the dinitrotol- chemicals were relatively nontoxic at the doses uene compounds. administered except DMH. which induced Histological examination of paraffin- 50% mortality when given po at a dose of 25 embedded tissues from MCA-treated mice in- mg/kg. dicated that the peritoneal tumors were poorly

When compared to the appropriate vehicle differentiated sarcomqs. These sarcomas were controls, MCA, DENA, urethan, and ENU spread throughout the peritoneal surface of produced significant (p < 0.05, t test) and the diaphragm, liver, stomach, pancreas, in- dose-related increases in the lung tumor re- testines, and kidneys in a circumferential sponse when given either ip or po. BP was growth that resembled the patterns seen in carcinogenic at all doses when given ip and mesotheliomas. However, only spindle-shaped at the highest dose after po administration. cells were observed in areas of parenchymal DMH was positive at the high and middle po invasion; epithelial-like cells forming pseudo- doses and at the highest dose following ip ad- glands typical of mesotheliomas were not seen. ministration. 2,4-DNT, 2,6-DNT, and the 2: 1 There were no grossly observable lesions mixture of 2,4-DNT and 2,6-DNT were in- other than lung adenomas in mice adminis- active by both routes and at all dose levels. tered ip DENA. However, in animals given

The tumor responses to MCA, BP, the po DENA, forestomach tumors were observed highest dose. of DENA, and the middle doses in 6 of 64 treated mice and liver lesions in 8 of both ENU and DMH varied as a function of 32 mice. The forestomach tumors were well- of the route of administration (Table 2). This differentiated squamous cell carcinomas that finding was most evident for the polycyclic varied from microinvasive papillary elevations hydrocarbons. At all doses, MCA and BP in- to invasive neoplasms that penetrated the duced significantly more lung tumors when stomach wall. The grossly observable liver le- given ip as compared to po. For example, the sions appeared to be the result of bile duct tumor response in animals receiving the mid- hyperplasia. The bile ducts appeared as mul- dle dose of MCA or the highest dose of BP tiple cystic profiles that occasionally exceeded by the ip route exceeded that by the po route 0.5 mm in diameter. A single neoplastic nod- by factors of 12 and 13, respectively. Only two ule was observed immediately under the cap- animals survived the highest ip dose of MCA sular surface of one liver. In addition, all eight (100 mg/kg); therefore, lung tumor responses livers contained multiple eosinophilic focal to 100 mg/kg MCA given either ip or po could areas of cellular alteration that were not ev- not be compared. DENA (200 mg/kg) and ident in the centrilobular portions of the liver. ENU (50 mg/kg) induced 1.45 (DENA) and Cells within the foci were two to three times 2 (ENU) times as many tumors per lung when larger than normal surrounding hepatocytes given ip as compared to po. In contrast, DMH, and their nuclei were pleomorphic, enlarged, at the dose of 12.5 mg/kg, produced signifi- and frequently contained nuclear inclusions. cantly more tumors per lung when adminis- A lymphosarcoma of the spine and an un- tered po. The lung tumor response to urethan differentiated carcinoma of the duodenum did not vary significantly by either route of were observed in mice given po ENU and administration. DMH, respectively.

Lesions in Other Organs Tissue Distribution 013-Melhylcholanthrene

Lesions other than lung adenomas observed After ip administration of 100 mg MCA/ at necropsy among the various chemically kg, the distribution of 3H material was ex-

an ad fec CO

tis 1 ;: thc at T t do as! pe an in! to H I6 in1 tht aft rac kic 0.7

LUNG ADENOMA BIOASSAY IN A/J MICE

TABLE 3

LESIONS OTHER T H A N LUNG TLJMORS

able 3. IS other he con- .itrotol-

araffin- nice in- : poorly as were face of eas, in- erential seen in -shaped ~chymal pseudo- .ot seen. lesions

dminis- Is given ~bserved Ins in 8 :re well- nas that :vations ~ted the liver le- ile duct as mul- xceeded ;tic nod- the c a p all eight lic focal not ev-

he liver. ee times atocytes nlarged, :lusions. 1 an un- odenum W and

rnthrene

g MCA/ was ex-

Number of animals Total dose with lesions/

Compound (mg/kg) Route number of animals Site H i s t o l o ~ c appearance

MCA"

MCA

BP

I BP

DENA

DENA

DENA

ENU

DMH

Peritoneal serosa

I Peritoneal serosa

Hind leg

Preputial gland

Forestomach

Liver

Forestomach

Spine

Duodenum

Sarcoma (poorly differentiated)

Sarcoma (poorly differentiated)

Fibrosarcoma

Adenocarcinoma

Squamous cell carcinoma

Bile duct hyperplasia

Squamous cell carcinoma

Lvmphosarcoma

Undifferent~ated carcinoma

' MCA. 3-methylcholanthrene: BP, benzo(a)pyrene; DENA, diethylnitrosamine; ENU. ethylnitrosourea; DMH. dimethylhydrazine.

amined in blood, urine. liver, lungs, kidneys, adipose tissue, small and large intestine, and feces. The total amount of radioactivity that could be accounted for in these fluids and tissues varied from 4.3 * 0.8% of the dose at 1 hr to 7.4 * 1.4% of the dose at 72 hr, with the majority (2.4 * 0.8% at 1 hr and 5.3 + 0.6% at 72 hr) present in the gastrointestinal tract. The remaining radioactivity (93-97% of the dose) could not be accounted for, but was assumed to be unabsorbed material in the peritoneal cavity. At all time points. the amounts of radioactive material in the large intestine, including that in the feces, were two- to fourfold higher than the total amount of 'H products excreted in the urine (Fig. 1). At 16 hr, the amount of 3~ products in the small intestine reached a maximum, while that in the large intestine and urine peaked at 48 hr after administration (Fig. 1). The contents of radioactive material in blood, liver, lungs, ludneys, and adipose tissue were low (0.1-0.3, 0.7-2.3, <O.l, 0.3-0.6, and 0.2-0.490 of the

dose, respectively) without clear evidence of changes with time. For liver and lungs. this result is illustrated in Table 4.

After po administration of 100 mg MCA/ kg, the total amount of 'H products that could be accounted for in fluids and tissues varied from 38.7 + 10.590 (8 hr) to 18.7 k 3.290 (72 hr) with intermediate values at the other time points. Most of this material was in the gastrointestinal tract and feces ( 1 4.6-36.9%) and the amounts present in the small and large intestines decreased over time (Fig. 2). Total amounts in urine were always lower than those in feces (30-fold at 8 hr, 4-fold at 72 hr). Total amounts of radioactive material in other organs (liver, lungs, kidneys, adipose tissue) and blood were very low and generally amounted to 30-5090 of the corresponding amounts observed after ip administration (Table 4).

Approximately 5090 of the amount of radioactive material present in the small intestine or liver 24 hr after ip administration rep- resented unchanged MCA (Fig. 3), while at

LUNG ADENOMA BIOASSAY IN A/J MICE

male A/J mice were used in transplacental studies. DMH, a strong colon carcinogen, has not been reported to induce lung tumors in strain A mice, and was only marginally active at the dose levels given in the present study. Currently, a repeat study with DMH is underway to determine if higher doses given over an 8-week period will elicit a stronger carcinogenic response. Hydrazine sulfate and 1.1- DMH induced lung tumors in BALB/c and SWR mice, respectively, when administered by gavage (Biancifiori and Ribacchi, 1962; Roe el al.. 1967). and at 200-fold higher doses than employed for I ,2-DMH in the present study. To our knowledge. this is the first report of the ability ofMCA, urethan. DENA, and ENU to induce lung tumors in strain A mice when administered po and of BP to induce lung

I 8 16 24 tumors when given ip. HOURS AFTER ADMINISTRATION The lung tumor responses to MCA, BP,

FIG. 3. Elimination of MCA (I00 mg/kg) from the DENA. ENU, and DMH were significantly liver, small intestme. and hngs. Strain A mice received different when the compounds were given ip an IP (A) or PO (8) dose of 100 mg MCA/kg as indicated to hi^ difference was par- in the legend to Fig. I. ['HJMCA was reisolated asdexribed under Methods. Each point represents the mean SD of t icular l~ evident for the P ~ ~ ~ ~ Y ~ ~ ~ ~ hydrocar- four mlce. bons, MCA and BP, which were several-fold

more active when given ip and indicates that MCA, BP, urethan. DENA, ENU, and the ip route is superior to the po route when

n to com- DMH induced a significant increase in the testing polycyclic hydrocarbons for lung tumor 1 series of lung tumor response when given both ip and induction in A/J mice. However, at high doses, a1 classes po. On a molar dose basis, the tumor responses strongly carcinogenic polycyclic hydrocarbons ne, nitro- to ip administered MCA and urethan were may induce a higher mortality when given ip 1 dinitro- similar to those previously reported in strain as compared to po (see data for 100 mg/kg A/J mice A mice (Stoner el al., 1973, 1974). BP, ad- MCA; Table 2). It is likely that the high mor-

ministered po, is approximately one-fifth as tality in mice given 100 mg/kg MCA ip was active for lung tumor induction in A/J mice due to the development of fibrosarcomas in (present study) than in A/HeJ mice (Watten- the peritoneal cavity since these tumors were berg and Leong, 1970). This result was un- observed in all seven mice (Table 3) necropsied

kg) expected since strains A/J and A/HeJ mice prior to death including the two animals that have similar lung tumor responses to ip ad- sunrived 24 weeks. Unfortunately, the other

- ministered urethan (Stoner et al., 1973). Pos- 25 mice that died during the bioassay were

- sibly the difference in tumor response is related too decomposed for necropsy; therefore, it is to the use of different vehicles [tricaprylin vs not known whether they had fibrosarcomas

:s corn oil (Wattenberg and Leong, 1970)J. or any other tumors.

0.07 DENA, given ip, was approximately four times Studies of the distribution and elimination 0.04 more active for lung tumor induction in A/J of MCA were undertaken to determine the 0.02 mice than in SWR (Swiss) mice (Mirvish and basis for the observed difference in the lung 0.0 1 0.01

Kaufman, 1970). The tumor response to ip tumor response when the compound is given

0.0 administered ENU is nearly identical to that either ip or po. These studies showed that low of an earlier report (Rice, 1969) in which fe- amounts of MCA (or its metabolites) are pres-

322 STONER ET AL.

ent in all organs, both after ip and po ad- Dinitrotoluene (DNT) compounds have ministration, and that these amounts are two- been tested for carcinogenic potential when to threefold lower after po than after ip administered in the diet. Technical grade DNT administration. A similar situation has been (75.8% 2,4-DNT, 19.5% 2.6-DNT, and 4.7% observed after ip administration of MCA to other isomers), when fed to F-344 rats at 3.5 rats (Black and Webster, 1974) and after po to 35 mg/kg/day, produced a dose-dependent

TI administration to hamsters (Mirvish et al., increase in hepatocellular carcinoma with a B U I L

1981). Elimination of MCA by A/J mice oc- higher incidence among males than among addt.

curs principally by the fecal route, with smaller females (CIIT, 1979). 2,6-DNT was classified scrir

amounts excreted in the urine (Figs. 1 and 2). as a "weak" hepatocarcinogen when fed to F- ron r CA 6

Similar results were obtained after intramus- 344 rats and exhibited promoting activity in Dcx ; cular administration to A/J mice (Dauben and rats initiated with a single dose of DENA Mabee, 195 1). While approximately 50% of (Leonard el al., 1982). 2,4-DNT (>98% pure) unchanged MCA is found in the liver and was also a hepatocarcinogen when fed to CD- small intestine 72 hr after ip administration, 1 rats at 34 and 45 mg/kg/day, and induced A?ir no unchanged MCA is found at this time after both preneoplastic and neoplastic renal tumors tr po administration. This finding suggests that in male CD-I mice when fed at 13.5 and 95 te

po administered MCA is extensively metab- mg/kg/day (Ellis el al., 1979). A study. con- B14'

olized in the gastrointestinal tract before being ducted by the National Cancer Institute, 11:

ta absorbed, although it is unknown how much showed no evidence for carcinogenicity of 2,4- B L . ~ unchanged MCA is eliminated from this or- DNT in either rats or mice (U.S.D.H.E.W., m

gan. Nevertheless, it appears possible that ei- 1977). We found that 2,4-DNT did not induce 3 '

ther less of the proximate or ultimate carci- lung tumors in A/J mice when given ip at Cti~

nogenic metabolite(s) of MCA is formed or doses of 3000, 1500, and 600 mg/kg (Schut ( 1 R

that this metabolite(s) is rapidly detoxified el al., 1982). In the present study, 2,4-DNT at (e.g., by conjugation) in the gastrointestinal was also inactive when administered po, and D41 tract. This result would lead to lower amounts confirms previous results on its lack of car- n

of the ultimate carcinogen reaching the lungs, cinogenic potential in mice (Schut et al., 1982). the target organ in the bioassay, and possibly Similarly, 2,6-DNT and the 2:l mixture of E L I

.I explain the lower tumor yield observed after 2,4-DNT and 2,6-DNT were inactive for lung I po administration (Table 2). Similar expla- tumor induction in A/J mice when given ip n

nations may apply to other polycyclic hydro- and po. The inability of these dinitrotoluenes E

carbons, such as BP, for which the lung tumor to induce lung tumors in A/J mice is not too P

response after ip administration was also much surprising since it is known that many he- c Hr '

higher than after po administration (Table 2). patocarcinogens are either inactive or only S

The lung tumor response to the highest dose weakly active for lung tumor induction in h

of DENA and the middle doses of ENU and strain A mice (Shimkin and Stoner, 1975; i .,

DMH also varied as a function of the route Maronpot el al., 1983). LF(

of administration. However, these differences In previous carcinogenicity studies, tumors 5

occurred at only one dose level and require were not observed on the surface of the liver I

2 verification by additional experiments. Ure- of strain A mice after the standard 6-month LF( than produced a similar lung tumor response bioassay, and histological studies of the liver \

when given by both routes. Pharmacokinetic were not performed (Shimkin and Stoner, c

studies of the uptake, distribution, and me- 1975). However, the identification of liver le- I

M. tabolism of urethan in A/J mouse tissues are sions in mice given DENA po in the present I underway to determine if the absence of a study suggests that the livers of strain A mice difference in tumor response after ip and po should be examined routinely for histopath- administration can be explained by phar- ological changes at the end of the 6-month macokinetic parameters. bioassay. This examination could permit the

ide~ ofa t1vc

LUNG ADENOMA BIOASSAY IN AIJ MICE 323

nds have tial when -ade DNT and 4.7% -ats at 3.5 lependent la with a .n among classified

I fed to F- lctivity in )f DENA 38% pure) ed to CD-

induced la1 tumors .5 and 95 udy. con- Institute, ity of 2,4- I.H.E.W., lot induce ven ip at kg (Schut 2,4-DNT d po, and :k of car- al., 1982). lixture of e for lung I gven ip otoluenes is not too many he- : or only uction in er, 1975;

:s, tumors f the liver 6-month

f the liver d Stoner, )f liver le- le present In A mice listopath- 6-month

e m i t the

identification of chemicals that produce foci MIRVISH, S. S., GHADIRIAN, P.. WALLCAVE. L.. RAHA,

of altered hepatocytes or other lesions sugges- c. . BRONCZYK, S., AND SAMS, J. P. (1981). ~ f f e c t of

tive of their carcinogenic potential. diet on fecal excretion and gastrointestinal tract distribution of unmetabolized benzo(a)pyrene and 3-meth-

ACKNOWLEDGMENTS ylcholanthrene when these compounds are administered orally to hamsters. Cancer Res. 41, 2289-2293.

The technical assistance of Diane Szmania. Cindy Butcher. and Tamara Gmitter is greatly appreciated. In addition, we thank Marilyn Cline for typing the manu- script. This research was sponsored jointly by the Envi- ronmenlal Protection Agency Cooperative Agreement No. CAB0767 1 and by the U.S. Army Medical Research and Development Command.

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