Chinese-German Journal of Clinical Oncology

December 2007, Vol. 6, No. 6, P533–P536

DOI  10.1007/s10330-007-0121-5

Development for the lung tumor model of newborn mice induced by diethylstilbestrol Yumei Zhang, Xia Sun, Jun Ying, Jinchun Pan Department of Veterinary Pharmacology, Veterinary College of Yangzhou University, Yangzhou 225009, China Received: 14 July 2007 / Revised: 30 July 2007 / Accepted: 27 August 2007 Abstract  Objective: To explore a economical and effective method for building lung tumor model induced by diethylstibestrol (DES). Methods: The carcinogenic effect of neonatal mice treated by DES was studied. The newborn mice were divided into DES, Urethan (U) and U + DES groups. U group was given in 500 mg/kg dose by ip at postnatal 14 day, DES group was administered by ip at the 1 d, 8 d, 15 d in the dose of 1/7, 2/7 and 4/7 LD50 of the day when they were injected respectively for DES (l), DES (M), DES (H) groups. Until 26 weeks, they were anatomized and checked the formation of tumors. The organ index, tumor incidence ratio and mean number of tumors were calculated. Results: Lung tumors were apparently induced in tested neonatal mice. The incidence of lung tumor of DES (L, M, H) groups were 16.7%, 22.4% and 43.1% respectively, the U + DES (L, M, H) groups were 70.4%, 90.9% and 70.8% respectively, and the U group was 53.1%. The mean numbers of lung tumors of U + DES (L, M) groups were higher than those of the DES (L, M) groups respectively (P < 0.05). Conclusion: The higher ratio of lung tumor incidence had been induced by DES and U joined action to neonatal mice, which may be a useful and economical method to establish a lung tumor model induced by DES. Key words  diethylstilbestrol (DES); newborn mice; tumor model; lung tumor

It has been very clear that sexual hormones play an important role in the genesis of cancers, such as carcinoma of endometrium and mammary cancer, etc., the relation of sexual hormones to the genesis of tumors in non-target such as liver, stomach and esophagus has also been reported [1–5]. There were more investigation to genesis of tumors in mice induced by diethylstilbestrol (DES) [6], mostly in long exposure to adult mice. The present study was conduced to observe the cancerogenesis effect of DES on new born mice in order to explore a economical and effective method for building lung tumor model.

Materials and methods Experimental animals and chemicals New born ICR mice, at postnatal 1 d, 8 d, 15 d old, were provided by the Center of Comparative Medicine of Yangzhou University (China) which the license number was SYXK (Shu) 2002-0045. DES was the product of the Shanghai Hualian Pharmaceutical Limited Company (China), batch number was 021002. Urethan (U) was come from the Shanghai Chemical and Reagent Company, the Medicine Company of China. U was dissolved in saline, Correspondence to: Yumei Zhang. Email: [email protected]

and DES was dissolved in some acetone and diluted with oil for injection. Mice were housed in a barrier facility, at a temperature of 20–25 ℃ and with a relative humidity of 50 ± 5% and a 12 h light/dark cycle. A basal diet and water were provided. The acute toxicity experiment to newborn mice The acute toxicity tests of 1 d, 8 d, 15 d old newborn mice exposed to DES by ip were conducted. The lowest dose were 10, 12, 15 mg/kg for these different birth time mice respectively, the highest dose were 18, 22, 25 mg/kg respectively. Six groups were divided at dose ratio in every birth time mice, the dose ratio was equal (the highest dose / the lowest dose) 1/5, the number of mice in every group was ten. The LD50 value was calculated according to the improved Kaber method, the formula was as below: LD50 = lq–1 [Xm – i (∑P – 0.5)] Sx50 = i × [(∑P – ∑P2) / (n – 1)]1/2 The 95% faith range of LD50 = lg–1 ( lgLD50 ± 1.96 Sx50) Xm: the logarithm of the highest dose; i: the logarithm of dose ratio; P: the decimal death ratio; n: the number of mice in a group; Sx50: standard error of the logarithm of LD50.

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Table  1  The programs of newborn mice treated by diethylstibestrol or/and urethan ip The day after born Groups 1st 8th 14th Control 0.85% salt water 0.85% salt water – Solvent DES’ solvent DES’ solvent – U – – 500 mg/kg DES (L) 1/7 LD50 (1 d) 1/7 LD50 (8 d) – DES (M) 2/7 LD50 (1 d) 2/7 LD50 (8 d) – DES (H) 4/7 LD50 (1 d) 4/7 LD50 (8 d) – U+DES (L) 1/7 LD50 (1 d) 1/7 LD50 (8 d) 500 mg/kg U+DES (M) 2/7 LD50 (1 d) 2/7 LD50 (8 d) 500 mg/kg U+DES (H) 4/7 LD50 (1 d) 4/7 LD50 (8 d) 500 mg/kg Table  2  The organ indexes of newborn mice treated by diethylstilbestrol or/and urethan (χ ± s, n = 10) Groups Liver Lung Uterus Control 5.04 ± 0.29 0.73 ± 0.09 0.89 ± 0.38 Solvent 5.05 ± 0.73 0.62 ± 0.10 0.79 ± 0.18 U 4.69 ± 0.22* 0.75 ± 0.15 0.68 ± 0.17 DES (L) 4.93 ± 1.04 0.61 ± 0.10* 0.30 ± 0.10* DES (M) 4.82 ± 0.53 0.63 ± 0.23 0.41 ± 0.11* DES (H) 4.56 ± 0.56* 0.61 ± 0.09* 0.36 ± 0.09* U + DES (L) 4.28 ± 0.66* 0.89 ± 0.76* 0.32 ± 0.09*＃ U + DES (M) 4.81 ± 0.80 0.68 ± 0.16 0.46 ± 0.14*＃ U + DES (H) 4.55 ± 0.52* 0.64 ± 0.15 0.63 ± 0.22*△

15th 0.85% salt water DES’ solvent – 1/7 LD50 (15 d) 2/7 LD50 (15 d) 4/7 LD50 (15 d) 1/7 LD50 (15 d) 2/7 LD50 (15 d) 4/7 LD50 (15 d)

Testicle 0.78 ± 0.23 0.64 ± 0.04 0.90 ± 0.03 0.72 ± 0.10 0.58 ± 0.16* 0.46 ± 0.15* 0.51 ± 0.17*△# 0.80 ± 0.13△ 0.45 ± 0.21*＃

Organ index = weight of organ (g) / body weight (g) × 100%; *, P < 0.05, compared with control; △, P < 0.05, compared with the same dose DES group respectively; ＃, P < 0.05, compared with the U group

Experimental methods and designation of groups The mice were divided randomly in nine groups, the control group was of saline, the solvent group was of DES’ solvent (some acetone dissolved and diluted with injection oil), and the treatment groups were the following: 500 mg/kg urethan group [sign U], the group of low dose of DES [DES (L)], medium dose of DES [DES (M)], high dose of DES [DES (H)], low dose of DES + U [U + DES (L)], medium dose of DES + U [U + DES (M)], and the group of high dose of DES + U [U + DES (H)]. The treatment programs about the dose of medicine and the time of intraperitoneal injection (ip) were list in Table 1. One hundred mice were in every group, males and females were separated to breed when mother milk was not used to feed. The death number were recorded within and out lactation periods. When 26 weeks old, the mice were dissected, pathological check was also made and the amount of liver, lung, kidney, uterus and testicle were weighted. The organ index was calculated in the formulae, organ index = weight of organ / body weight of mouse. At the same time, the lung was fixed with formaldehyde, all pieces of tumor and normal lung tissue were, respectively, taken out for routine paraffin slices, and the slices were subjected to hematoxylin eosin staining, then observed by microscope.

Statistical analysis The comparisons of mouse body weights and average amount of tumors between groups were made by t test, and the comparisons of incidences of tumors between different groups were made by χ2 test.

Results The LD50 value of neonatal mice administered by DES The LD50 values of different time of mice at 1 d, 8 d, 15 d old treated by DES were LD50 (1 d) = (13.9 ± 0.9) mg/kg, LD50 (8 d) = (18.4 ± 0.8) mg/kg, LD50 (15 d) = (20.8 ± 0.6) mg/kg respectively. Effects of DES and/or urethan on the organ index of mice The organ indexes of mice in control and solvent groups were not different from each other, therefore, the statistical assay was compared with the control group. The uterus indexes in every DES and U + DES groups were lower than that of the control group (P < 0.05). The testicle indexes in the groups of DES (M), DES (H), U + DES (L), and U + DES (H) were obviously lighter than that of the control (P < 0.05). Comparing with the control group, the liver index in U group was lower (P < 0.05), but other organ indexes were not different from the control

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Chinese-German J Clin Oncol, December 2007, Vol. 6, No. 6 Table  3  The incidence rate of lung tumor and mean numbers per mouse treated by diethylstilbestrol or/and urethan Groups

No. of mice dissected

No. of mice with lung tumor

No. of lung tumors

Incidence rate (%) Mean No. of lung tumors

Control 55 0 0 0.0 0 Solvent 61 1 1 1.6 1.00 ± 0.00 U 64 34 113 53.1* 3.33 ± 0.85* DES (L) 60 10 25 16.7* 2.50 ± 1.20* DES (M) 67 15 40 22.4* 2.67 ± 0.32* DES (H) 58 25 88 43.1* 3.52 ± 0.68* U + DES (L) 71 50 239 70.4*△# 4.78 ± 1.72*△# U + DES (M) 66 60 254 90.9*△# 4.23 ± 0.95*△# △# U + DES (H) 48 34 125 3.68 ± 1.23* 70.8* Incidence rate = No. of mice with lung tumor / No. of mice dissected ×100%; Mean No. of lung tumors = No. of lung tumors / No. of mice with lung tumor; *, P < 0.05, compared with control; △, P < 0.05, compared with the same dose DES group respectively, ＃, P < 0.05, compared with the U group Table  4  The rate of mice death between the experiment periods Groups

No. of mice tested

Until the time weaning No. of mice

The death rate A (%)

When be dissected No. of mice

The death rate B (%)

Control 100 62 38.0 55 11.3△ Solvent 100 63 37.0 61 3.2△* U 97 74 23.7 64 13.5△ DES (L) 99 64 35.4 60 6.3△* DES (M) 100 75 25.0 67 10.7△ DES (H) 98 66 32.7 58 12.1△ U + DES (L) 100 83 17.0 71 14.5△# U + DES (M) 99 76 23.2 66 13.2△ U + DES (H) 98 53 45.9 48 9.4△ The death rate A = (No. of mice tested – No. of mice when be weaned) / No. of mice tested, The death rate B = (No. of mice when be weaned – No. of mice when be dissected) / No. of mice when be weaned. *, P < 0.05, compared with control; △, P < 0.01, compared with the rate of death A in the same group; ＃, P < 0.05, compared with the same dose DES group respectively

(P < 0.05). Comparing with U group, the uterus and ovary indexes in the groups of DES (L) and DES (M) were lower, the testicle indexes in the groups of U + DES (L) and U + DES (H) were also lower (Table 2). Pathological examination of the lung tumors By the direct observation and the use of the anatomical microscope, lung tumors in DES, U + DES and U groups were identified, and with diameters of 0.1–3 mm. The tumors were beneath the pulmonary pleura and prominent slightly on the surface of lungs. Pathological observation under the microscope showed the lung tumors were mainly adenomas, slightly papillomas. The tumors in liver or ovary were also seen, with stripeless or gobbet in liver tumors and adenocarcinomas in ovary. The incidence rate of lung tumor and mean numbers per mouse in the U, DES or U + DES groups were different from that in the control (Table 3). Effects of DES and/or U on the death rate of mice The death rates (A and B) of mice were respectively calculated at the periods of drinking milk and during the

time from ablactation to the date be dissected. The statistics analysis (χ2 test) were conducted for the death rate B when mice were dissected (Table 4).

Discussion The lung tumors were significantly occurred when ICR mice were only administrated by DES and/or urethan for three or fore times. The incidence rates of lung tumor of mice treated by U + DES groups were higher than 70%. The effects of DES and/or urethan on newborn mice lung tumorigenesis were effectively used for the development of lung tumor model, which increased work efficiency and consumed litter manpower compared with the effect of DES/urethan on adult mice lung tumor by injection once every week for 18 weeks [6]. The experimental results showed more cancer risk in infancy period of animal or man exposure to DES-like carcinogens. Current models of DES-related neoplasia follow the initiation-promotion paradigm of carcinogenesis. The promotional properties of endogenous or pharmaceutical estrogens on uterine, cervical, vaginal or breast cancers are well established; however, the roles of genotoxic, epi-

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genetic or hormonal actions of estrogens or DES in tumor initiation remain uncertain [7]. Numerous studies have illustrated the induction of DES-like effects in laboratory animals following exposure to various other pharmaceutical, industrial or naturally occurring xenoestrogens [8, 9]. Therefore, further characterization of the DES model is certain to advance our knowledge of the potential carcinogenic and developmental risks associated with human exposure to a variety of xenoestrogens. The term perinatal is used refer broadly to preconceptional, gestational, and infancy periods. Human exposures during this period could contribute to two distinct types of risk: of cancer in children and young adults, and of cancer later in life. These two scenarios could be expected to be qualitatively different, because childhood cancers, with short latency, often contain molecular lesions that may have been present at birth, whereas neoplasms that appear in middle or old age, as a result of perinatal events, more likely would reflect chronic conditions and often collaboration with other causative factors [10]. In addition, many of the more common childhood cancers are rare in adults, and vice versa.

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