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[CANCER RESEARCH 52, 1948-1953. April I, I992|

252CfRelative Biological Effectiveness and Inheritable Effect of Fission Neutrons inMouse Liver Tumorigenesis1

Tadateru Takahashi, Hiromitsu Watanabe, Kiyohiko Dohi, and Akihiro Ito2

Department of Cancer Research, Research Institute for Nuclear Medicine and Biology [T. T., H. W., A. I.], and Second Department of Surgery, School of MedicineÂ¡K.D.], Hiroshima University, Kasumi 1-2-3, Minami-Ku, Hiroshima 734, Japan

ABSTRACT

To determine the relative biological effectiveness of 252Cffission neutrons versus *Â°Co7-rays for inducing liver tumorigenesis, C57BL/6NCrj

x C3H/NOJ F,, hereafter called B6C3F,, mice were irradiated onceeither with 252Cfor "Co and the tumorigenicity of the two types of

radiation was studied.Individual groups of B6C3F] mice (about 30 mice/group) were irradi

ated once with 252Cfat doses of 0, 3, 6, 12.5, 50, and 200 cGy for malesand 0, 12.5, 50, and 200 cGy for females or with "Co at doses of 0, 12.5,

50, and 200 cGy for both sexes. The groups were observed for 13 monthsafter irradiation.

The incidence of hepatic tumors in nonirradiated controls was 3.8% inmales and 3.2% in females. 252Cfirradiation increased the incidence dose

dependent!) in males and females, but less efficiently in females. Themean number and size of hepatic tumors were well correlated with tumorincidence. In contrast, ''"Co was less efficient at inducing tumors. Therelative biological effectiveness of 2S2Cfcompared to MCo was quite highin the low-dose range. Overall, the average relative biological effectiveness of 252Cfversus MCo was 15.2 in males and 2.5 in females.

In another experiment, 7-week-old male C3H mice, which are knownto be carriers of hepatic tumorinogens, and that were the fathers of theB6C3F, mice, were irradiated with 0, 50, and 200 cGy of 252Cf,and 2

weeks later they were mated with nonirradiated female C57BL mice. Thecontrol mice had nonirradiated fathers. The incidence of hepatic tumorsin the offspring of irradiated fathers was 43% in males and 1.7% infemales, whereas in the offspring of control mice with nonirradiatedfathers the incidence was 3.2% in males and 3.3% in females. Themultiplicity of the hepatic tumors was similar to the tumor incidence.

Thus our results indicated a strong hepatocarcinogenic potential for252Cfneutrons compared to *Â°Co7-rays, and that the cancer-prone genetictrait activated by 252Cfirradiation was inherited by first-generation off

spring, especially in males.

INTRODUCTION

Hepatic tumors occurring in C57BL/6NCrj x C3H/NCrj F,,hereafter called B6C3F,, mice are a suitable model for assessingthe carcinogenicity of various physiological or environmentalfactors (1, 2). Except for the endogenous factors in this strainof mice that influence its hepatotumorigenicity, such as sex,nutrition, and age at the time of administration of the carcinogen (3, 4), radiation seems to be one of the most powerfulagents in inducing hepatic tumors in B6C3F, mice. 252Cfis aman-made isotope that spontaneously emits a mixture of neutrons and 7-rays in a 67:33 ratio, with half-lives for the twoprocesses of 2.6 and 85.5 years, respectively (5, 6). It is alsoused as a neutron source in radiotherapy against malignantdiseases (7, 8). In the rÃ©Ã©valuationof the dosimetry and consequent biological effectiveness of atomic bomb radiation in Hiroshima and Nagasaki (the Dosimetry System 1986 [DS86])the role of neutrons received major attention (9). Follow-up

Received 6/24/91; accepted 1/27/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Mice Â»eremaintained under the guidelines set forth in the "Guide for theCare and Use of Laboratory Animals" by Hiroshima University.

2To whom requests for reprints should be addressed.

studies of the atomic bomb survivors at the Radiation EffectsResearch Foundation (10) have shown that they have a relativerisk greater than 1.0 for various neoplasms, including leukemia,multiple myeloma, colon, breast, and lung tumors. Currently,there is much interest in the relative biological effectiveness offission neutrons in tumorigenesis, especially at low dose levels(11). As for the effectiveness of radiation in inducing tumors inmouse liver, DiMajo et al. (12) reported a RBE3 for fission

neutrons of 28 at 9 cGy for prenatal irradiation and 13 at 17cGy for irradiation at 3 months of age in male B6C3F, mice.There has already been a report of the late effect of fast neutronsand 7-rays in mice, especially in inducing various types ofneoplasms (13).

In this study we designed experiments to observe directly thecarcinogenic effects of 252Cfneutrons and 60Co 7-rays in miceand the inheritability of 252Cf the effect on the induction of

neoplasms, including hepatic tumors.

MATERIALS AND METHODS

RBE for Hepatic Tumorigenicity

Animals. COBS B6C3FÂ¡mice of both sexes were purchased fromCharles River Japan, Inc. (Kanagawa, Japan). About six mice werehoused together in autoclaved cages with sterilized wood chips, andwere kept in a room with controlled temperature (24 Â±2Â°C)and

humidity (55 Â±10%) under a regular 12-h light, 12-h dark cycle. All ofthe mice were given a normal diet (Oriental Co., Ltd., Tokyo, Japan)and tap water ad libitum.

252CfNeutron Irradiation. A 252Cfsource (71.4 GBq) at the Research

Institute for Nuclear Medicine and Biology, Hiroshima University, wasused for neutron irradiation. The emitted radiation from the 252Cfsource consisted of 67% fission neutrons and 33% 7-rays. Neutron and7-ray doses were measured by using ion chambers (1C-17 and IC-17G;Far West Technology, Goleta, CA) and Fricke and thermoluminescentdosimeters (6). The average energy of 252Cfneutrons is 2.13 MeV (14).Six-week-old B6C3Fi mice were exposed to whole-body irradiation ina ferris wheel irradiator (Chiyoda Safe Products Co., Ltd., Tokyo) at adose rate of 0.6-0.8 cGy/min. Individual animals were put into acrylatemesh containers that allowed free movement within the space. Theferris wheel was rotated slowly during irradiation. All irradiation procedures were carried out at room temperature.

The mice were divided into the following groups. Group 1 consistedof male mice divided into six subgroups according to radiation dose:Group la (n = 55), 0 cGy; Group Ib (n = 24), 3 cGy (the pure neutrondose = 2.0 cGy); Group le (n = 24), 6 cGy (3.9 cGy); Group Id (n =32), 12.5 cGy (8.1 cGy); Group le (n = 32), 50 cGy (32.5 cGy); andGroup If (n = 32), 200 cGy (130 cGy). Similarly, Group 2 consistedof female mice divided into four subgroups: Group 2a (n = 66), 0 cGy;Group 2b (n = 32), 12.5 cGy; Group 2c (n = 32), 50 cGy; Group 2d (n= 32), 200 cGy. The irradiation times were approximately 5 min for 3cGy, 10 min for 6 cGy, 21 min for 12.5 cGy, 83 min for 50 cGy, and333 min for 200 cGy.

MCo 7-Ray Irradiation. Six-week-old mice were exposed to whole-body 7-ray irradiation from a 60Co source at a dose rate of 1.0 cGy/

min. Each of 10 mice were kept in square acrylate mesh containers that

3The abbreviations used are: RBE, relative biological effectiveness; HCS,hepatocarcinogen-sensitive locus.

1948

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2"Cf FISSION NEUTRONS IN MOUSE LIVER TUMORIGENESIS

allowed free movement within the space during irradiation. The dosim-etry was similar to that described for 2"Cf. The mice were divided intotwo groups in the same manner described for 2"Cf irradiation. Group3 consisted of male mice, divided into three subgroups: Group 3a (n =30), 12.5 cGy; Group 3b (n = 32), 50 cGy; and Group 3c (n = 27), 200cGy. Similarly, Group 4 consisted of female mice divided into threesubgroups: Group 4a (n = 29), 12.5 cGy; Group 4b (n = 32), 50 cGy;and Group 4c (n = 27), 200 cGy.

Inheritable Effects of Neutron Irradiation

Animals. C3H/NCrj male and C57BL/6NCrj female mice werepurchased from Charles River Japan, Inc. Seven-week-old C3H malemice were exposed to whole-body 252Cfirradiation with total doses of

0, 50, and 200 cGy, and 2 weeks later they were mated with unexposedC57BL females. After mating, vaginal smears from 10 C57BL mice ofeach group were examined every morning to determine the exact dateof fertilization. On the 18th day of gestation, pregnant mice weresacrificed under ether anesthesia and intact and aborted embryos werecounted. About 20 pregnant C57BL females were examined in a similarmanner, but were delivered naturally and the pups were weaned whenthey were 4 weeks old.

Pathology. All mice were observed every day and weighed once amonth. They were sacrificed 13 months after irradiation in ExperimentA and at 14.5 months of age in Experiment B. Mice were autopsiedunder ether anesthesia, and their body weights and the weights ofvarious organs were measured. The number and size of the liver tumornodules were also measured. Diseases of the liver and most organsincluding those with neoplastic changes were subjected to routinehistolÃ³gica! studies. Liver tumors were stained with hematoxylin andeosin, periodic acid-Schiff, or the van Giesson method. Some micereceived s.c. injections of iron dextran (Tekiraiser, Pfizer Co., Ltd.,Tokyo) at the dose of 0.125 mg/g body weight 3 or 4 days before theywere autopsied (15). Iron-positive Kupffer cells were present in hyper-

plastic areas, but not in neoplastic areas (15, 16). Liver nodules werehistologically classified as neoplastic or nonneoplastic lesions, following the criteria of the "Pathology of Tumors in Laboratory Animals"

(17). Briefly, they were mainly classified according to the findings oftumor size, an increased number of trabecular cords, a well-defined

border, signs of compression in the surrounding parenchyma, variationsin cell morphology, and the absence of triads.

Statistical Analysis. The x2 test and Student's t test were used fordata analysis. The RBE of 252Cfbased on '"Co was calculated by using

the single-hit model.

RESULTS

Experiment A

Survival and Body Weight. The survival rates at 13 monthsafter 252Cfor 60Co irradiation are shown in Table 1. More than

95% of the control mice survived the experimental period. Thesurvival rates of 200 cGy 252Cf-irradiated mice were 91% in

both sexes, and thus no acute fatal effect was evident for thisirradiation. A preliminary study showed that the median lethaldose of "2Cf was about 400 cGy in B6C3F, mice. Average body

and liver weights and the liverbody weight ratios are also shownin Table 1. In the 252Cf-irradiated groups, the body weights of

Groups Ib and Id were significantly higher than that of Groupla, and that of Group If was significantly lower than that ofGroup la. Average liver weights, in general, changed in accordance with the change in body weights. The average body weightsat the end of the experiment for both 252Cf-and M>Co-irradiated

mice were always significantly increased compared to those ofunirradiated controls for both sexes except in Groups If and2d. However, the liverbody weight ratios were decreased forboth sexes in the irradiated groups compared to those of therespective unirradiated controls.

Tumors. Table 2 shows liver tumor and total tumor incidences. In Group la there were 3.8% liver tumors and 1.9%pulmonary tumors. In Group 2a there were 3.2% liver, 1.6%pulmonary, pituitary, lymphoma, and 4.8% ovarian tumors. In252Cf-irradiated mice in both sexes there is a positive correlation

between the incidence and spectrum of tumors and the radiationdose. Tumor multiplicity was also correlated with the radiationdose. In 60Co irradiation in Groups 3 and 4 there was also a

positive correlation between tumor incidence and radiationdose, although tumor incidence was lower in comparison tothose of the neutron-irradiated groups.

Incidence of Liver Tumors. Tumor incidence, the number oftumors per mouse, and mean tumor size for each group areshown in Table 3. The incidence of liver tumors in male micewas always higher than that in female mice for every dosegroup. There was a clear dose-related correlation of tumorincidence, the number of tumors, and mean tumor size for 252Cf

irradiation in both sexes. Especially at low doses (3, 6, and 12.5cGy), there was an abruptly increased incidence of liver tumors

Table I Experimental design, survival rate, and average body and liver weights

IrradiationGroup

SexTypelacÃ®":CfIbleIdleif2a

ÃŽ"2Cf2b2c2d3a

i"Co3b3c4a

9"Co4b4cDose

(cGy)03612.550200012.55020012.55020012.550200Effectiveno. ofmice53242430302963293029302925293126Survival

rate(%)961001009494919591949110091931009796Body

wt(g)Start24.52.1.723.324.423.525.218.919.020.020.024.223.022.419.318.818.9End42.5

Â±4.1Â°45.8Â±3.0*41.9

Â±5.744.2Â±2.8C43.4

Â±3.238.6Â±5.0*36.3

+6.140.9Â±5.7*43.3Â±7.1*38.0

Â±9.245.2

Â±4.0*46.5Â±3.7*45.8Â±3.6*36.9

Â±4.745.2Â±6.6*46.9Â±7.9*Liver

wt(g)2.09

Â±0.352.26Â±0.24r1.98

Â±0.471.78Â±0.47*2.05

Â±0.301.91Â±0.45C1.47

Â±0.191.90 Â±0.66*1.46

Â±0.251.73Â±0.47*1.75

Â±0.26*1.99

Â±0.272.13+0.251.32

+0.25*1.53

Â±0.231.59+ 0.33Liver

wt asmean %

of bodywt4.94

Â±0.754.96Â±0.554.72Â±0.884.01

Â±0.42*4.72

Â±0.514.89Â±1.164.1

3Â±0.694.65Â±1.503.39Â±0.34*4.37

Â±0.743.85

Â±0.37*4.28Â±0.51*4.05Â±0.51*3.59

Â±0.36*3.42Â±0.52*3.59+

1.67' Means Â±SD.* Significantly different from control, P < 0.01 (by / test).' Significantly different from control. P < 0.05 (by t test).

1949



2"Cf FISSION NEUTRONS IN MOUSE LIVER TUMORIGENESIS

Table 2 Incidence of tumors

Effettivino. of

GroupmicelaIbleIdleIf2a2b2c2d3a3b3c4a4h4c53242430302963293029302925293126Total5.720.825.043.346.779.312.713.823.369.013.317.244.003.223.1Incidence

of tumors(%)Liver3.812.520.836.743.362.13.23.46.727.63.317.236.00015.4Lung1.94.28.3013.301.63.403.43.300000Adrenal0003.30003.46.720.73.300003.8Skin0003.306.9003.310.3000000Ovary4.8288062382377Others04.2"00010.3*3.2C3.4'6.7'lo.y6.6'08.0*03.2'3.8"Multiplicity(%)Double004.2010.013.806.9013.8000000Triple0000000003.4000000

" One forestomach papilloma.' One kidney tumor, one gastric adenoma, and one lymphoma.' One pituitary adenoma and one lymphoma.d One lymphoma.' Two harderian gland tumors.^One pituitary adenoma and two harderian gland tumors.* One bladder tumor.* One lymphoma and one harderian gland tumor.1One thymoma.

Table 3 Incidence of hepatocellular tumors

GrouplaIbleIdleIf2a2b2c2d3a3b3c4a4b4cEffectiveno. ofmice53242430302963293029302925293126Incidence

ofliver

tumors(%)3.812.5Â°20.8Â°36.7f43.3r62.1f3.23.46.727.6f3.317.2e36.(r"0015.4CMeanno. of

tumors/mouse0.040.130.250.400.601.210.030.030.070.340.030.170.44000.15Mean

tumorsize

(mm)0.510.88

Â±0.56*1.96

Â±0.942.37Â±0.742.97

Â±0.616.26Â±0.740.210.070.371.03

Â±0.440.101.66

Â±0.862.10Â±0.63000.62

Â±0.36* Significantly different from control. P < 0.05 (by x2 test).* Values are means Â±SE.' Significantly different from control. P < 0.01 (by x2 test).

in male mice. Moreover, all incidences in the individual dosegroups were much higher for 2"Cf irradiation compared tothose for 60Co. The RBE of 252Cfbased on 60Co was calculatedby using the single-hit model, and the maximum likelihoodestimates were 15.2 in males (Fig. 1) and 2.5 in females (Fig.2).

Pathology. Radiation-induced liver tumors were rather lowin malignancy and had low cellular dysplasia. In contrast to theprevalent occurrence of eosinophilic tumors induced by chemical carcinogens, most of the hepatic tumors obtained in thepresent study were basophilic, as is also observed in spontaneous liver tumors. Their transplantability into syngeneic micewas not more than 35% in both sexes (data not shown). Nometastasis was noted to other organs. In this experiment,altered foci were separately scored and were not included in

neoplastic disease. In iron-dextran injected mice, both normalliver tissues and altered foci showed the diffuse presence ofKupffer cells loaded with phagocytized iron. However, in neoplastic tissues, it was clearly shown that neoplastic areas weredevoid of iron-positive cells because those iron-loaded Kupffercells do not exist in these areas (Figs. 3-6).

Experiment B

1. Effect of Neutron Irradiation on Embryos. These observations were made on the 18th day of gestation. Pregnancy rateswere 50% in the 0-cGy group, 40% in the 50-cGy group, and20% in the 200-cGy group. Survival rates were 93.5% at 0 cGy,76.5% at 50 cGy, and 33% at 200 cGy, thus showing a dose-dependent decrease in survival due to increased intrauterinefetal death (Table 4).

2. Tumor Incidence in Offspring of Irradiated Fathers. Table

12.5 50 100 150Irradiation (cGy)

200

Fig. 1. Comparative incidence of liver tumors in male B6C3F, mice exposedto 2!2Cfneutron ( ) and To -y-ray radiation ( ). The RBE of ";Cf basedon *Â°Cois 15.2. using the single-hit model.

12.5 50 100 150Irradiation (cGy)

200

Fig. 2. Comparative incidence of liver tumors in female B6C3F, mice exposedto 2!2Cfneutron ( ) and MCo -y-ray radiation ( ). The RBE of 252Cfbasedon *Â°Cois 2.5. using the single-hit model.

â€”¿�',>;:-- *-v:-/.A.-..- vrÃv.,

Â«5Â»-'?$Â£:-:â€¢ 'S Â¿

â€¢¿�&yÃ²iÃ¬:OÂ±<:i-

Fig. 3. Basophilic cell foci in the liver of a male mouse after 200 cGy of "2Cfneutron whole-body irradiation. Note the marked increase of vacuolated cells andthe basophilic granular cytoplasm of hepatic cell foci. H & E.

1950



2!!Cf FISSION NEUTRONS IN MOUSE LIVER TUMORIGENESIS

Fig. 4. The same lesion as in Fig. 3. Iron-phagocytized Kupffer cells are presentat the hepatic cell foci. Iron staining.

Fig. 5. A hepatocellular tumor in a male mouse 13 months after 200 cGy of:!2Cf neutron whole-body irradiation. Note the disorganized liver cell structureand the well-defined tumor border. H & E.

of offspring in the group whose male parents were irradiatedwith 50 cGy of 252Cfwas 44 males and 58 females versus 30

males and 31 females in the nonirradiated control group. Thebody weights of the offspring of irradiated fathers were significantly higher than those of the controls.

Table 6 shows the incidence of hepatic tumors observed inB6C3F! mice. The frequency of hepatic tumors in the offspringof nonirradiated males was 3.2%, but it increased to 43.2% inthe offspring of irradiated males. The number of tumors permouse and mean tumor size were similar to the tumor incidence. There was no difference in tumor incidence between thefemale offspring of irradiated and nonirradiated fathers.

The footnote to Table 6 gives the incidences of various tumorsother than liver. In females, ovarian tumors were the mostcommon, followed by pituitary tumors. Only liver tumorsshowed a correlation with 252Cfirradiation.

DISCUSSION

There are reports that radiation can induce liver tumors incombination with carbon tetrachloride (18) or partial hepatec-tomy (19). The present experiments indicated that fission neu-

Table 4 Effect of "2Cfirradiation on embryos

Dose(cGy)050200Embedded78754b35366Intrauterinedeath00200211044Pregnancyrate(%)504020Â°Survival

rate(%)93.576.5Â°33.3Â°

1Significantly different from control. P < 0.01 (by x! test).

.

/ I

Table 5 Average body and liver weights of offspring in Experiment BFather's2!2Cfdose(cGy)Â°050050Sex

ofoffspringMaleFemaleNo.ofmice31443058Average

wt(g)Body4I.9Â±

3.6*45.1Â±5.3C33.0

Â±4.246.2Â±6.2CLiver2.3

Â±0.3*2.3

Â±0.41.

5Â±0.21.7Â±0.3Liver

wtasmean%of

bodywt5.4Â±0.6*5.0

Â±0.74.6

Â±0.43.7Â±0.4

Â°Male mice were exposed to whole-body 2"Cf irradiation, and 2 weeks later

were mated with intact female C57BL mice.* Values are means Â±SD.r Significantly higher than those of controls. P< 0.01 (by x! test).

Table 6 Incidence of liver tumors in offspring in Experiment B

Fig. 6. The same lesion as in Fig. 5. Iron-phagocytized Kupffer cells are notpresent in the neoplastic area. Iron staining.

5 presents the experimental groups, number of mice examinedin each group, average body and liver weights, and the livenbodyweight ratio at the termination of the experiment. The offspringwhose male parents were irradiated with 200 cGy of 252Cfdid

not survive more than 2 days after birth. The effective number

Father'sdose

(eGy)0

50050Sex

ofoffspringMaleFemaleNo.ofmice31

44Â°30e58*Incidence3.243.2"3.3

1.7Liver

tumorsNo.

oftumors/mouse0.03

0.910.03

0.02Mean

size(mm)0.32

3.750.10

0.07" Incidence of other tumors: pituitary, 2.3%; thyroid. 4.5%; and adrenal, 2.3%.* Significantly different from controls. P < 0.01 (by x2 test).r Incidence of other tumors: adrenal. 3.3%; ovarian, 20%.d Incidence of other tumors: pituitary, 5.2%; adrenal, 1.7%; and ovarian, 8.6%.

1951



"!Cf FISSION NEUTRONS IN MOUSE LIVER TUMORIGENESIS

iron or 7-ray irradiation alone could induce liver tumors in adose-dependent manner in juvenile B6C3F, mice without anyfurther additive treatments. There are many reports concerningchemical carcinogenicity in the liver in relation to enzymemetabolism (20, 21). However, there are very few reports onradiation-induced liver tumors dealing with radiation doses orthe tumorigenic RBE of 252Cfcompared to X-rays (12). This

may be because the liver, unlike, for example, lymphoid, thyroid, thymus, or mammary tissues (22), has not been considereda direct target organ for radiation carcinogenesis.

In this study we terminated the experiments 13 months afterirradiation because spontaneous liver tumors start to appear inB6C3F, mice around 14 months of age. There are reports ofspontaneously occurring liver tumors in B6C3F, mice in which21.6% of males and 3.9% of females developed tumors over a90- to 110-week observation period (23), and 33.2% of malesand 1.6% of females developed tumors over a period of 166weeks (24). Thus, irradiation both shortened the latency andincreased the incidence of liver tumors. Therefore, based onthis information and the results of a preliminary experiment,we felt that it was wise to observe tumor incidence before thespontaneous tumors began to occur.

The present results imply that radiation could be effectivenot only at the initiation stage, but that it may also act as apromoter for the occurrence of liver tumors. It should also bestated that the liver tumors observed in the present study arehistologically quite similar to spontaneous tumors, but they areclearly different from the tumors induced by chemical carcinogens (25-27).

There was a clear relationship between the doses of both the2"Cf and 60Co irradiation and the frequency of liver tumors.Especially with low-dose 252Cfirradiation in the range of 0 to

12.5 cGy, there was an abrupt increase in tumor incidence.Although there is a contamination of 7-rays into the neutroncomponent, and its influence should be considered in the assessment of biological effectiveness, it is considered that thebiological effect of the y component in the range of 0 to 12.5cGy total dose would be negligibly small because of its lowproductivities of hepatic tumor incidence, and therefore noattempt was made to subtract the y contribution from the totaleffect. Maruyama et al. (28-30) evaluated the RBEs of fissionneutrons as measured by the loss of weight in the testis, thymus,and spleen relative to the RBEs of 60Co or 137Cs7-rays, andfound that they were 3.7, 1.47, and 1.0-1.9, respectively. Withers et al. (31) reported that the RBE of fission neutrons (252Cfcompared to radium 7-rays) was less than 5 for killing ofintestinal crypt cells. Sato et al. (32) and Shigeta et al. (33) inour institute reported that the RBE of 252Cfwas around 2 to 3for teratogenicity and sensitivity to colony-forming units ofhematopoietic stem cells. It is calculated that the RBE of 252Cfbased on 60Co for hepatic tumorigenesis is as high as 15.2 inmale mice and 2.5 in female mice. This high RBE for hepato-tumorigenicity is in accordance with the value of 13-28 reportedby DiMajort al. (12).

In human beings a sex difference in liver tumor incidence hasbeen well delineated. In Japan the ratio between males andfemales was about 2:1 in 1970, and the incidence in males issteadily increasing (34). In B6C3F, mice there is also a clearsex difference, with a higher incidence in males than in females.This finding is further clarified by the finding that the incidenceof hepatic tumors was significantly decreased in gonadecto-mized males.4 The incidence of chemically induced liver tumors

4T. Takahashi et al., manuscript in preparation.

has also been reported to be decreased by gonadectomy (35). InCBA mice the incidence of hepatic tumors was 16-29% inmales and 3-5% in females over a 15- to 30-month observationperiod. In the same study the incidences were 12-55% in maleand 5-11% in female C3H mice (36). Similar observations havealso been reported (37). As to the sex difference of otherradiation-induced tumors, B6WF, mice were reported to havea prevalence of pituitary and adrenal tumors in females causedby X-rays (38).

In Experiment B there is strong evidence that some geneticconstituents for cancer-prone traits could be transmitted intothe next generation through neutron-irradiated sperm. Onereport notes that sperm-shape abnormalities were observed inmice exposed to 252Cfradiation (39). However, we must remem

ber that C3H mice are known to spontaneously develop livertumors, predominantly in males. Drinkwater and Ginsler (40)named the gene responsible for this trait the "hepato-carcinogensensitive locus." The HCS has a role in the sensitivity to hepatic

tumorigenesis in hybrids of C57BL and C3H mice. In ourexperiments, the activation of the HCS by neutron irradiationmay be a causative factor for the increased incidence of hepatictumors in F, offspring. Again, it should be emphasized that theincidence of hepatic tumors was higher in males than in females.This finding could be explained by a combined influence of theHCS and sex differences. The strain of C3H/HeN mice usedin the present study are known to have a high incidence ofmammary tumors as well as liver tumors. In the inbreedingprocess, however, mouse mammary tumor virus was artificiallyabrogated in the progenitor by having the newborn pups fed bynon-mouse mammary tumor virus carrier C57BL mice. However, there is as yet no evidence that a hepatic tumor virus, if itexists, might be biologically potent (41). It is highly suggestivethat both sexes of B6C3Fi mice could have a gene encodingHCS contaminated by a hepatic tumor virus, and that this viruswould be fully amplified in males but would not be expressedin females.

Nomura (42) previously reported that the incidence of pulmonary tumors in urethan-treated ICR mice was greatly increased in the offspring of X-ray-irradiated parents comparedto those of nonirradiated parents. The administration of ethyl-nitrosourea to male rats similarly resulted in a high incidenceof neurogenic tumors in their offspring (43). Thus, it may bethat carcinogenic insults to male rodents are being efficientlytransmitted into their offspring. There are some reports indicating that the activation of RAS or erb-B genes could explainthe phenomenon of the genetic transmission of carcinogenictraits (44, 45). Further study will be needed to clarify this issue.

ACKNOWLEDGMENTS

The authors would like to thank Professor S. Sawada for valuablediscussions, Dr. M. Ohtaki for performing the statistical analysis ofRBE, and S. Fukuma and S. Uemukai for their technical assistance.

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1992;52:1948-1953. Cancer Res Tadateru Takahashi, Hiromitsu Watanabe, Kiyohiko Dohi, et al. Fission Neutrons in Mouse Liver Tumorigenesis

Cf Relative Biological Effectiveness and Inheritable Effect of252

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