[CANCER RESEARCH 55, 1271-1276, March 15, 1995] Cancer Induction by an Organic Arsenic Compound, Dimethylarsinic Acid (Cacodylic Acid), in F344/DuCrj Rats after Pretreatment with Five Carcinogens1
Shinji Vaniamolo,2 Yoshitsugu Konishi, Tsutomu Matsuda, Takashi Murai, Masa-Aki Shibata, Isao Matsui-Yuasa,3 Shuzo Otani, Koichi Kuroda, Ginji Endo, and Shoji Fukushima First Department of Pathology ¡S. Y., Ts. M., Ta. M.. M-A. S.. S. F.1, Second Department of Biochemistry //. M-Y., S. O.j, and Department of Preventive Medicine and Environmental Health /K K., G. E.I, Osaka City University Medical School, 1-4-54 Asahi-machi, Aheno-ku, Osaka 545, and Osaka Cit\ Institute of Public Health and Environmental Sciences, Osaka 543 IK. K.], Japan
ABSTRACT Taiwan and Mexico are exposed to high amounts of As via the drinking water (5, 8). Moreover, the wide population in the United Arsenic (As) is environmentally ubiquitous and an epidemiologically States may be supplied with water containing more than 50 ju.g/1As significant chemical related to certain human cancers. Dimethylarsinic (6). Industrial arsenicals are used for smelting, glass making, and the acid (cacodylic acid; DMA) is one of the major methylated metabolites of manufacture of semiconductors (3, 9). For more than half a century, ingested arsenicals in most mammals. To evaluate the effects of DMA on chemical carcinogenesis, we conducted a multiorgan bioassay in rats given various carcinogenic effects of As for humans have been documented, various doses of DMA. One-hundred twenty-four male F344/DuCrj rats mainly involving the skin and lung (7). In addition, recent epidemi- were divided randomly into 7 groups (20 rats each for groups 1-5; 12 rats ological studies have indicated that there are significant dose-response each for groups 6 and 7). To initiate multiple organs and tissues, animals relationships between inorganic As ingestion and cancer incidences of in groups 1-5 were treated sequentially with diethylnitrosamine (100 the kidney, urinary bladder, liver, and other internal organs (5, 6). mg/kg body weight, ¡.p.,single dose at the commencement) and \ -inclÃnI- DMA is a major form of As in the environment (1) as well as being V-nit rosónITU (20 mg/kg body weight, ¡.p.,4 times, on days 5, 8, 11, and used as a general herbicide or pesticide for many years (4). Further 14). Thereafter, rats received 1,2-dimethylhydrazine (40 mg/kg body more, DMA is one of the major methylated metabolites of ingested weight, S.C.,4 times, on days 18, 22, 26, and 30). During the same period, the animals were sequentially administered .V-lmt>l-.Y-(4-lmini\jl>ii- organic (10) or inorganic (11) As in most mammals, including humans tyl)nitrosamine (0.05% in the drinking water, during weeks 1 and 2) and (3, 4, 12, 13), and is eliminated by the kidney and finally excreted in V-l>isl2-liy
Received 10/6/94; accepted 1/18/95. The cosls of publication of this article were defrayed in part by the payment of page MATERIALS AND METHODS charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely lo indicate this fact. 1This work was supported by a Grant-in-Aid for Cancer Research from the Ministry Animals. Male F344/DuCrj rats were obtained at 5 weeks of age from of Education, Science and Culture of Japan. Charles River Japan, Inc. (Hiño,Japan). Rats were divided randomly into 7 2 To whom requests for reprints should be addressed. groups (20 rats each for groups 1-5; 12 rats each for groups 6 and 7), were 3 Present address: Department of Food and Nutrition, Faculty of Human Life Science, housed 5/sIeel cage, and fed common basal diet MF (Oriental Yeast Co., Ltd., Osaka City University, Osaka 558, Japan. 4 The abbreviations used are: As, arsenic; DMA, dimethylarsinic acid; ODC. ornithine Tokyo, Japan) and water ad libitum. The animals were kept at a temperature of decarboxylase; SAT, spermidine/spermine W'-acetyltransferase; DEN, diethylnitro 23 ±1°Cwith a 12-h light/dark cycle. After a 1-week acclimation period, they samine; DMH, 1,2-dimethylhydrazine; BBN, A'-butyl-A'-(4-hydroxybutyl)nitrosamine: were used in this study. MNU, /V-methyl-iV-nitrosourea; DHPN, /V-bis(2-hydroxypropyl)nitrosamine; DMBDD Chemicals. DEN and DMH were purchased from Wako Pure Chemical model, DEN-MNU-BBN-DMH-DHPN model; GST-P, glutathione S-transferase placenta! Industries, Ltd. (Osaka, Japan), BBN from Tokyo Kasei Kogyo Co., Ltd. form; PAPG, pepsinogen 1 (Pgl) altered pyloric glands: MMA, monomethylarsonic acid; TMA, trimethylarsine oxide; As'", arsenous acid; IC-ICP-MS, inductively coupled (Tokyo, Japan), and MNU and DHPN from Iwai Chemical Co. (Tokyo, Japan). plasma mass spectrometry with ion chromatography; TCC, transitional cell carcinoma. DMA was purchased from Wako Pure Chemical Industries (purity 99%). 1271
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. DIMETHYLARSINIC ACID ENHANCEMENT OF CARCINOGENESIS
Experimental Design. The experimental protocol of this bioassay system method. Sections were then counterstained with hematoxylin for microscopic is shown in Fig. 1. To establish wide-spectrum initiation of organs and tissues, examination. The numbers and areas of GST-P-positive foci in the liver animals in groups 1-5 were treated sequentially with DEN (100 mg/kg body sections and the length of pyloric mucosa in each histological slide for weight, i.p., single dose at the commencement of the experiment) and MNU quantitative analysis of PAPG were measured with the aid of a color video (20 mg/kg body weight, i.p., 4 times, on days 5, 8, 11, and 14). Thereafter, rats image processor (VIP-21 C; Olympus-Ikegami Tsushin Co., Tokyo, Japan.). received DMH (40 mg/kg body weight, s.c., 4 times, on days 18, 22, 26, and Areas of bilateral renal cortex sections stained with hematoxylin and eosin 30). During the same period, the animals were sequentially administered BBN were also measured for quantitative analysis of the putative preneoplastic (0.05% in the drinking water, during weeks 1 and 2) and DHPN (0.1% in the lesion, atypical tubules (29), by image analyzer. drinking water, during weeks 3 and 4; DMBDD treatment). After a 2-week Measurement of ODC and SAT Activities and Polyamine Biosynthesis. interval, groups 2-5 were given 50, 100, 200, or 400 ppm DMA (27.1, 54.3, Two enzymatic activities, ODC and SAT, as biomarkers of cell proliferation in 108.6, and 217.1 ng/ml as As), respectively, in the drinking water. These kidneys and lungs were measured by the methods of Matsui et al. (30) and DMA concentrations were selected based on previous experimental data (24). Otani et al. (31), respectively. The animals without DMA administration Group 1 received basal diet and tap water without any chemical supplement (group 1) served as controls. DMBDD plus 100 ppm of DMA (group 3) was after the DMBDD treatment. Groups 6 and 7, which were not given DMBDD chosen for evaluation. At the time of terminal sacrifice, frozen pieces of rat treatment, received 100 and 400 ppm DMA during weeks 6-30. Throughout kidney and lungs were suspended in 0.5 ml of 50 mM Tris (pH 7.5) containing the experiment, all rats had free access to food and water, and body weights 0.25 M sucrose and disrupted by 10 strokes with a Potter-Elvehjem homoge- were measured each week. At week 10, fresh urine was collected by forced nizer. The homogenized suspensions were centrifuged at 100,000 X g for 30 urination, immediately frozen, and stored until use for measurement of As min, and the supernatant was assayed for ODC and SAT activity by measure concentrations. All survivors were killed by exsanguination under ether anes ment of the amount of radioactive putrescine produced from [5-'4C]ornithine thesia at week 30. and the amount of acetyl moiety transferred from [l-'4C]acetyl-CoA to sper- Tissue Processing and Histopathological Diagnosis. All major organs midine, respectively. Some frozen kidney tissues were used for the measure were excised and fixed in 10% buffered formalin except for the forestomach ment of three polyamines. They were homogenized in 4 volumes of ice-cold and glandular stomach. After adequate fixation, the nose of each rat was 5% trichloroacetic acid and centrifuged for removal of protein. The concen removed from the head, trimmed of extraneous tissue, and decalcified for trations of polyamines in the acid extract were analyzed by HPLC (Shimadzu approximately three days. They were sectioned into three levels for further LC-6A; Shimadzu, Kyoto, Japan) equipped with a fluorescence detector. processing as described by Young (25). All organs and tissues excised were Concentration and Speciation of As in Urine. Using the urine samples embedded in paraffin and stained with hematoxylin and eosin for histological collected at week 10, five As species, MMA, DMA, TMA, arsenobetaine, and examination. Liver slices fixed in ice-cold acetone and stomach slices in As"1 were analyzed with the aid of IC-ICP-MS (32, 33). We applied ion sublimated formaldehyde were embedded in paraffin and used for quantitative chromatography (Model 1C 7000; Yokogawa Analytical Systems, Inc., Tokyo, assessment of putative preneoplastic lesions, GST-P-positive foci in the liver Japan) with two anión exchange columns (Excelpak ICS-A35; Yokogawa (26) and PAPG in the glandular stomach (27). Frozen tissues of both kidneys Analytical Systems) as the separation technique and ICP-MS (Model PMS and both lungs were used for measurements of enzymatic activity of ODC and 2000; Yokogawa Analytical Systems) for detection. Briefly, urine samples SAT and concentrations of three kinds of polyamines. were diluted 10-fold, and 50 /j.1were used for assessment. For separation of the Examination of GST-P-positive Foci in the Liver and PAPG in the five As species, 1C was operated under the following conditions: mobile phase, Glandular Stomach. We evaluated preneoplastic lesion markers for assess 10 HIMtartane acid; flow-rate, 1.0 ml/min; and column temperature, 50°C.The ment of carcinogenic activity, i.e., immunohistochemically demonstrated GST- eluent of the ion chromatography column was directly introduced into the P-positive foci in the liver (26) and PAPG in the glandular stomach (27). The nebulizer of the ICP-MS and analyzed at monitoring mass 75. liver tissues in groups 4 and 5 were not évaluablefor preneoplastic lesions Statistical Analysis. The significance of differences between mean values because tumor induction was so pronounced that the tumors occupied most of was analyzed using Student's / test. The significance of differences in lesion the livers. In groups 2 and 3, materials containing liver tumors (hyperplastic incidence between groups was assessed by Fisher's exact probability test. nodule or hepatocellular carcinoma) were excluded from the evaluation. For Two-tailed Cochran-Armitage analysis was used to evaluate the dose-response the assessment of PAPG, stomach tissues in groups 1, 3, and 5 were used. relationships for lesion incidences in groups 1-5. For the assessment of mean Sublimated formaldehyde for fixation of the stomach was made from 6% values of preneoplastic lesions (GST-P-positive foci, PAPG, and atypical HgCU in a mixture of 5% acetic acid and 4% formaldehyde. The avidin-biotin- tubule), the one-tailed Dunnett multiple comparison procedure was used. peroxidase complex method (28) was used to determine the location of GST-P binding in the liver and Pgl binding in the pyloric mucosa. After deparaf- finization, the liver and glandular stomach sections were treated with rabbit anti-rat GST-P (1:5,000) and rabbit anti-rat Pgl (1:10,000), respectively. The RESULTS sites of peroxidase binding were demonstrated by the diaminobenzidine Survival and Main Cause of Death. In DMBDD-treated groups (groups 1-5), only seven animals were found dead or killed during the 30 Wks experiment. One animal of group 2 given 50 ppm DMA and three -Ih animals of group 3 given 100 ppm DMA died of thymic lymphoma during weeks 24-27. One animal of group 1 (control group) died at week 29 because of occlusion due to an esophageal cardia sarcoma. In groups 6 and 7, which were noninitiated but treated with DMA, all .DMA adminislralion (50.100.200.400 ppmi j rats survived for 30 weeks and maintained a relatively healthy ap pearance throughout the experiment. All rats were considered in the S 6.7 r DMA administration (100.400 pçm); O (12) L effective number with one exception in Group 3. Some organs of one animal in group 3 were not évaluablebecause of postmortem changes. : DEN f : MNU 7 : DMH :BBN : DHPN Body Weight and Relative Organ Weight. During the DMBDD I : DMA •:sacrifice treatment, body weight gain in the treated groups was less compared Fig. 1. Experimental protocol of multiorgan carcinogenesis bioassay (DMBDD mod to that of nontreated rats. We observed moderate body weight sup el). Animals were sequentially treated with DEN (arrow. 100 mg/kg body weight, i.p., pression by DMA at week 10; conversely, mean body weights became single dose), MNU (T, 20 mg/kg body weight, i.p., 4 times, on days 5, 8, 11, and 14), DMH (V, 40 mg/kg body weight, s.c., 4 times, on days 18, 22, 26, and 30), BBN (I. higher in the high-dose groups at the end of the experiment (groups 1 0.05% in the drinking water, during weeks 1 and 2), and DHPN (•.0.1% in the drinking versus 3 and 4; P < 0.01; Student's t test). In the tumor-bearing water, during weeks 3 and 4). After a 2-week interval, groups 2-5 were given 50, 100, 200, or 400 ppm DMA, respectively, in the drinking water (0). All survivors were killed organs, relative organ weights of liver and kidneys were higher in at week 30 (•). DMA high-dose groups; however, in the lungs and thymus, relative 1272
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. DIMETHYLARSINIC ACID ENHANCEMENT OF CARCINOGENESIS organ weights were decreased as higher amounts of DMA were given Macroscopically, most bladder tumors induced by DMA were (data not shown). whitish and showed papillary growth. Bladder calculi were not ob Water Intakes and the Concentrations of As in Urine. Water served in any groups. Neoplastic urinary bladder-epithelial lesions intake values (average of measurements made at weeks 12, 18, 24, were divided into papillomas and TCCs (34). In DMBDD-treated and 30) in group 5 were about 2 times higher than in the control animals, urinary bladder carcinogenesis was markedly enhanced by group 1 (P < 0.001). Higher amounts of water intake and DMA DMA, even in the lowest dose group (number of tumor-bearing intake were related to DMA doses. In groups 1-5, concentrations animals, group 1 versus 2;P< 0.001; Table 1). A preneoplastic lesion, of DMA in urine samples were 0.11, 5.4, 20.0, 35.4, and 54.7 papillary or nodular hyperplasia (34), was significantly observed in all jig/ml as As, respectively. Proportions of DMA in the total As DMBDD plus DMA groups. We observed four invasive TCCs in the content in urine of groups 1-7 were 49, 22, 46, 59, 59, 40, and higher DMA groups (each two animals of groups 4 and 5), one 56%, respectively. Although the main As metabolite in urine squamous cell carcinoma arose within TCC, and one carcinoma in situ samples was DMA as described in most previous reports (10), the in the same animal of group 5. In groups 6 and 7 (without DMBDD proportion of TMA was relatively high, and less As was excreted treatment and DMA-treated groups), no tumor was observed, as well as inorganic As (As111) in this experiment (data not shown). The as no preneoplastic lesions. amount of MMA was smaller, and arsenobetaine was not detected The numbers of kidney tumors (adenomas and/or adenocarcino- in any groups. mas) were significantly increased in a dose-dependent manner (Table Histopathological Examination and Tumor Incidences. Neo 1; P < 0.001; two-tailed Cochran-Armitage analysis). All renal ade plasms were found in several organs, suggesting a specific organot- nomas and adenocarcinomas were basophilic and solid. But all car rophy of DMA. Histopathology and tumor incidences in various cinomas were well differentiated, noninvasive, and revealed no evi organs are indicated in Table 1. In DMBDD-treated groups (groups dence of metastasis to other distant organs. No tumor was observed in 1-5), DMA significantly enhanced the tumor induction in the urinary groups 6 and 7. Typical toxic lesions in the renal cortex, such as bladder, kidneys, liver, and thyroid gland. massive tubular necrosis, were not commonly observed, even in the
Table 1 Incidences of preneoplastic and neoplaslic lesions in various organs from rats that received DMBDD treatment
Dx"UrinaryOrgan and (n =20)4(20)1(5) (n =20)13 (n =19)'14 (n =20)11(55)"1 (n =20)11(55)"7(35)* Cochran-Armitageanalysis'NENE bladderPN hyperplasiaPapilloma* (65)'12(60/ (73.7/12 (63.2/ 1(55/ TCC*No. 1(5) 10(50)' 11(57.9/ 12 (60/ 13 (65/ NE animalsKidneyAdenoma*-**Adenocarcinomaof tumor-bearing 2(10)1(5)01(5)4(20)5(25)10(50)1(5)1(5)000003(15)2(10)1(5)3(15)8/19*17(85/3(15)03(15)03(15)12(60)2(10)2(10)02(10)002(10)4(20)1(5)1(5)2(10)3/1816Õ84.2/1 17(85/7(35)*1(5)8(40)'6(30y16(80)'3(15)7 NEP
(5.2)2(10.5)2(10.5)4(12.1)'6(31.6)14 *Renal *-* (35)'-*9 <0.01P celltumorNephroblastoma*No. (45)'-'9 <0.001P A19 (45)>13 <0.05P animalsLiverAlteredof tumor-bearing (65)*-*20(100)/:''17(85rJ16 <0.001P
cellfociClear cellfociBasophilic (73.7)3(15.8)8(42.1)'-'2(10.5)0002(95)'-1110 <0.001P fociEosinophilic (50f*15 <0.001P fociHyperplastic (7$yj9 (SO/77 <0.001P nodule*HCC*Cholangioma*Hemangioma*No. (4Syj8 (35)**8 <0.001P (40)'-'1(5)1(5)17(85)"13(40)'-'1(5)013 <0.001P
animalsThyroidof tumor-bearing (10.5)2(10.5)3(15.8)5(26.3)8(42.1)'3/13(23.1)1/13(7.7)1/13(7.7)1/13(65yj13 <0.001P glandHyperplasiaAdenoma*Adenocarcinoma*No. (OS)''*1(5)5(25)6(30)7/20(65)'-"6 <0.001P (30)'4(20)9
animalsNasalof tumor-bearing (45)*1'4/19(21.1)00020(100)9(45)00Two-tailed<0.05P cavityPN hyperplasiaPapilloma/adenoma*Carcinoma*No.(42)3/19(15.8)2/19(10.5)5/19(16.6)1/18(5.6)1/18(5.6)2/18(11.1)20(100)5(25)1(5)0Group'13(35)2/20(10)02/20(10)20(100)6(30)1(5)1(5)5
animalsLungHyperplasiaAdenomaAdenocarcinomasec1of tumor-bearing (26.3)'20(100)10 (7.7)19(100)10 <0.05NENENENE
(50)01(5)2 (52.6)1 (5.3)04
" Dx. diagnosis; PN, papillary or nodular; HCC. hepatocellular carcinoma; SCC. squamous cell carcinoma. Hematoxylin and eosin-stained sections are marked with (*) and are totaled in "No. of tumor-bearing animals." **, totaled in "Renal cell tumor." * Groups 1, 2, 3, 4, and 5: 0, 50, 100, 200, and 400 ppm DMA. respectively. Percentages are in parentheses. ' Some organs of one animal in group 3, which died of thymic lymphoma, were not évaluablebecause of postmortem changes. **The significance of differences in lesion incidence between groups was assessed using Ihe Fischer's exact probability lest. To evaluate the dose-response relationships of lesion incidences in the kidney, liver, thyroid gland, and nasal cavity, two-tailed Cochran-Armitage analysis was used. NE. not examined. *~* Significantly different from group 1 at * P < 0.05, ' P < 0.01, and//> < 0.001. *-•'Significantlydifferent from group 2 at 'P < 0.05, * P < 0.01, and> P < 0.001. No. of lesions/effective nasal cavity specimens. Significantly different from group 5 at P < 0.05. 1273
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. DIMETHYLARSINIC ACID ENHANCEMENT OF CARCINOGENESIS highest DMA groups (groups 5 and 7). Atypical tubules, which have B a basophilic cytoplasm and occasionally contain granules, have been 200 100 100 regarded as a preneoplastic lesion in the renal cortex (29). Numbers of 01 atypical tubules/cm2 renal cortex significantly increased with DMA E 2000 administration in a dose-dependent manner (group 1 versus 5; TT •50 P < 0.001; one-tailed Dunnett multiple comparison procedure; Table 100 50 2). The data for atypical tubules in groups 6 and 7 [groups 6 1000 (0.11 ±0.27; mean ±SD) and 7 (0.14 ±0.18)] were lower than for group 1. Immunohistochemically demonstrated GST-P-positive liver cell Kidney Lung 0 Kidney Lung ODC SAT foci, a putative preneoplastic lesion, have been assessed as a marker for rat liver carcinogenesis (26). The moderate dose of DMA admin istration following DMBDD treatment (100 ppm; group 3) signifi 100 20 100 cantly enhanced the mean numbers and areas per unit area (cm2) of
GST-P-positive foci compared to the DMBDD-alone group (group 1 o DMBDD only (Group 1) ot versus 3; P < 0.001; Table 2). Neoplastic and preneoplastic lesions —Ü— 50 10 diagnosed on the hematoxylin and eosin sections were classified into 50 DMBDD plus DMA (Group 3) categories as listed in Table 1. There were dose-response relationships between the given dose of DMA and occurrence of altered cell foci (P < 0.001). High incidences of hyperplastic nodules and hepatocel- lular carcinoma were observed in rats administered more than 200 Putrescine Spermidine Spermine I kidney ' ppm DMA (Table 1), usually bearing more than two tumors per liver. Most carcinomas were solid or trabecular; other types (papillary or Fig. 2. Two enzymatic activities, ODC (A ) and SAT (H). in the kidneys and lungs, and polyaminc concentrations in the kidneys (C). ODC and SAT activities and three poly- tubular) were rare. Liver tissues in DMA-treated rats without amines were measured as described in "Materials and Methods." Tissues from rats without DMBDD retained normal features without evidence of toxicological DMA administration (group 1) were used for controls. Data are expressed as means ±SD (bars). Means with an asterisk are significantly different; *. P < 0.001; **, P < 0.01, changes, except for one basophilic focus observed in group 7. Student's t test. Follicular hyperplasia, which contains rich colloids and has bal looning follicles with atypical epithelial cells, was commonly ob served in high DMA-treated groups (Table 1). The incidences of both squamous cell carcinoma of the cheek and one s.c. fibroma on the follicular and C-cell adenoma/adenocarcinoma in DMA-treated back were present in the DMBDD plus DMA groups (group 2). groups were significantly higher than in the control group (group 1 The alteration of Pgl, disappearing or preferentially decreasing in versus 5; P < 0.05; Table 1). Solid and poorly differentiated adeno- areas of rat pyloric mucosa, can be immunohistochemically detected carcinoma was the most common type in the high DMA dose groups. as PAPO during the early stages of stomach carcinogenesis (27). Colloidal follicles of the thyroid gland appeared essentially normal. Numbers of PAPG/cm of mucosal length significantly increased in There was no remarkable pathological changes in the animals of groups 3 and 5 [group 1 (4.76 ±2.2: mean ±SD) versus group 3 groups 6 and 7. (11.0 ±3.1); P < 0.001; group 1 versus 5 (10.1 ±3.8); P < 0.001: Contrary to the positive effects of DMA described above, tumor one-tailed Dunnett multiple comparison procedure]; however, no in- induction of the nasal cavity tended to be suppressed by DMA (group tergroup difference was observed between the DMBDD plus DMA 1 versus 5; P < 0.05; Table 1). Most of the tumors arose from the groups (groups 3 and 5). transitional epithelium and had both papillary and inverted growth No remarkable histopathological changes were observed in other patterns. organs, even in high-dose DMA-treated rats. Overall, no métastases Lung, Skin, and Other Organs. All rats which received the were observed for any tumors in any of the groups, and no neoplastic DMBDD treatment developed alveolar hyperplasia of the lung change was observed in rats without DMBDD treatment (groups 6 and (Table 1). In addition, there were no other remarkable pathological 7), despite the relatively large amounts of DMA. changes in DMA-treated lungs. We could find no evidence of pro ODC and SAT Activities and Polyamine Biosynthesis. As moting effects of DMA on respiratory carcinogenesis. Pathologically, shown in Fig. 2A, ODC activities of the kidney tissue in DMBDD plus no remarkable changes were observed in the skin. Only one sebaceous 100 ppm DMA-treated rats were about three times higher than in DMBDD-only controls (P < 0.001). Kidney SAT activity did not differ between groups (Fig. 2B). Corresponding to these results, the Table 2 Quantitative tinta for prt'tit'oftlaxtii' lesion* //( AvW/K'viintlliver concentrations of two polyamines, sperminc and spermidinc, in the kidney tissues were significantly higher in group 3 compared with Kidney Liver control values (Fig. 2C). ODC and SAT activities in the initiated lung GST-P-positive foci tissues displayed no increase with oral administration of DMA (Fig. 2, Atypical tubules DMBDD DMA Effective (no./cm2 Numbers Areas A and B). Group treatment (ppm) no. of rats renal cortex) (no./cm2) (mm2/cm2) ±0.73*0.37 12345+0+ ±1.93.37 ±0.240.330 DISCUSSION 50+ ±0.500.45 ±1.36.03 ±0.250.654 100+ ±0.331.02 +2.9dNENE0.367 +0.27'NENE 200+ ±0.48''1.26 Despite the numerous attempts to induce tumors by administration 40020/20"20/1818/1620/-20/-0.46+ 0.76''3.50 of DMA using experimental animals, no adequate data have been " No. of specimens; kidney/liver. published (2, 18). Only a few reports using inorganic As compounds * Mean ±SD. are available (35-37). Johansen et al. (18) suggested that DMA might ' NE. not examined because liver tumors occupied most areas of the specimens. ''Significantly different from Group 1 at P < 0.001 (One-tailed Dunnett multiple be a promoter in rat kidney and liver carcinogenesis. The present comparison procedure). study showed that DMA has significant enhancing effects on 1274
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. DIMETHYLARSINIC ACID ENHANCEMENT OF CARCINOGENESIS carcinogenesis, not only in the kidney and liver, but also in the after oral administration; however, concentrations of DMA in rat urinary bladder and thyroid gland. Urinary bladder carcinogenesis thyroid gland have not been described. was strongly enhanced by the oral administration of DMA, even at The carcinogenicity of As in human lung and skin have been the lowest concentration (50 ppm; group 2) used in this experi reported (7), and previous reports using experimental animals de ment. Tumor inductions of kidney and thyroid gland were moder scribed a relatively high accumulation of ingested DMA in the lung ately enhanced by DMA in a dose-dependent manner. Strong and skin (10); however, we did not observe any enhancement of tumor enhancement of tumor induction in liver was observed in groups induction by DMA in these two organs. The biological markers ODC administered more than 200 ppm. and SAT activities in lung revealed no enhancing effect of cell The urinary bladder has long been described as a major target organ proliferations by the oral administration of DMA. The modulating of chemical carcinogenesis, probably related to the excreted proxi effect of DMA in the rat nasal cavity seemed quite opposite to that in mate metabolites of ingested chemicals being kept in the urinary humans, for which increase in cancer of this type has been described bladder for relatively long duration with continuous exposure to the in areas of endemic As exposure (5). This discrepancy is not satis urothelium. In numerous reports about human exposure to inorganic factorily clarified by our rat experiment, however, because the route As (3, 4, 9, 12, 13) and now corroborated by our data, DMA is the of exposure in humans is more complicated and the rat nasal cavity is major excreted metabolite of ingested As. In addition, it has been histologically and biologically different from humans. Considering reported that most of the absorbed DMA is excreted as unchanged the route of exposure for humans, further attempts to study respiratory DMA in the urine of various species (4, 10). Thus, we can conclude carcinogenesis (including nasal cavity) of As in laboratory animals that DMA itself may act as a urinary bladder carcinogen or particu should be done by intratracheal instillation or inhalation using inor larly as a potent promoter. Moreover, most kinds of ingested As ganic As (36, 37). No specific statement concerning skin carcinoge compounds must have a high possibility for enhancing urinary bladder nicity can be made since specific initiation of skin is not included in carcinogenesis, although combination treatment with other chemicals our model. No remarkable induction of tumors was observed in the having initiating activity might be required. gastrointestinal tract, with the exception of preneoplastic lesions in the The kidney is the major route of elimination of ingested As from glandular stomach. Further examination will be required concerning the human or animal body (10-12), and most of the metabolites are As carcinogenicity of the skin as well as the gastrointestinal tract since methylated, such as DMA, MMA, or TMA. Most reports of the some epidemiological studies have revealed an association of As with metabolism of ingested As show accumulation of DMA in the kidney tumors in these organs (5-7). (10). Mineral toxicology studies have focused particularly on proxi Cell proliferation has long been regarded as playing an important mal tubular alterations. Murai et al. (24) earlier described renal role in each stage of chemical carcinogenesis (40) and is now thought lesions, such as massive tubular necrosis and regeneration, induced by to be reflected partially by two enzymatic activities, ODC and SAT high-dose administration of DMA to F344 rats. The P3 segment of the (22, 23). We observed significantly high activity of ODC in the proximal tubule has been regarded as a frequent site of toxic injury in DMA-treated kidney. Our biochemical data indicate significant cell F344 rats (38), and Murai et al. (24) suggested that DMA affected this proliferation in 100 ppm DMA-treated kidneys, although tumor de area. In our results, however, renal tubular change was rare, even at velopment was low. Cohen and Ellwein (40) suggested that an in the highest DMA dose, although most of the renal cell tumors pro crease in cell proliferation can account for the carcinogenicity of duced by DMA presented a basophilic and solid phenotype, and these nongenotoxic compounds; however, the actual mechanism of induc are thought to be derived from proximal tubules (29). Aoki et al. (39) tion of cell proliferation by DMA is still unknown. also focused on these areas and revealed alteration of in vitro protein It is generally accepted that carcinogens have both initiating and synthesis in primary cultures of rat kidney proximal tubule cells by promoting activities and promoters have promoting activity without exposure to arsenite. The numbers of atypical tubules in kidneys of initiating activity (40, 41). In the present medium-term bioassay, we groups 6 and 7 were lower than those of group 1. However, they are demonstrated promoting activity of DMA in carcinogenesis in four conceivably higher than those of normal kidneys, and it remains a internal organs. However, considering the clastogenic (15, 16) or possibility that cancer induction by DMA would occur in long-term genotoxic (16, 17) activity of DMA, it may thus act as a carcinogen experiments without carcinogen pretreatment. Although nephroblas- by inducing DNA strand breaks or as a promoter acting through other tomas, similar to Wilms' tumors in humans, have been regarded as mechanisms for these organs. spontaneous neoplasms in this bioassay system without any signifi For the purpose of evaluating any carcinogenic effects of chemical cance, a relatively high incidence of this tumor was observed in the substances, various bioassay systems have been established (19, 20, high DMA dose groups. 26, 27, 29). Recently, rat multiorgan carcinogenesis models have been Quantitative data for preneoplastic lesions of the liver, numbers and developed for the detection of carcinogens (19, 20) and have already areas of GST-P-positive foci, and numbers of eosinophilic foci in the proven useful for this purpose. For example, Hirose et al. (21) 100 ppm DMA group were significantly increased compared to those reported that NaNO2 can enhance carcinogenesis when applied with of controls. Significant liver tumor induction was observed in rats certain phenolic antioxidants, particularly in the forestomach of rats. treated with 200 ppm DMA or more. These data suggest that 100 ppm The lack of any possible interaction with carcinogen initiation allows DMA might have some enhancing effect in liver carcinogenesis. effective assessment in a wide range of target organs (19). The results There have been several studies of the pharmacokinetics and me of such studies can be confirmed using various kinds of biomarkers, tabolism of isotope-labeled DMA in various species (10) and are i.e., preneoplastic lesions or indications of cell proliferation as used in helpful in interpreting the data from our multiorgan experiment. the present study. Accumulation of DMA occurs particularly in the kidney, liver, and There have been numerous reports concerning the possible carci maybe in the thyroid. In humans, thyroid gland cancer induction by nogenicity of As in limited areas on the southwest coast of Taiwan (5) As has not been emphasized in most epidemiológica! studies (5, 6); and elsewhere (6, 8). Artesian well water with a high concentration of however, moderate enhancement of thyroid neoplastic lesions was inorganic As has been used for more than 60 years in Taiwan, and significantly observed in the 400 ppm DMA group of this study. An these areas are known as having a high prevalence of some kinds of in vivo study by Vahter et al. (10) using mice revealed late-phase internal cancers. Our data are consistent with these epidemiological retention of isotope-labeled DMA, particularly in the thyroid gland findings. Both inorganic and organic ingested As are methylated in the 1275
Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. DIMETHYLARSINIC ACID ENHANCEMENT OF CARCINOGENESIS body and mainly excreted in urine as DMA, although this methylation 17. Yamanaka, K., Hasegawa, A., Sawamura, R.. and Okada, S. Cellular response to process had been believed to be a detoxification mechanism for oxidative damage in lung induced by the administration of dimethylarsinic acid, a major metabolite of inorganic arsenics, in mice. Toxicol. Appi. Pharmacol., 108: inorganic As (10-13). 205-213. 1991. In summary, ingested As may act as a carcinogen or promoter in 18. Johansen, M. G., McGowan, J. P., Tu, S. H., and Shirachi, D. Y. Tumorigenic effect of dimethylarsinic acid in the rat. Proc. Wesl. Pharmacol. Soc., 27: 289-291, 1984. these four organs: urinary bladder, kidney, liver, and thyroid gland. 19. Ito. N.. Shirai, T.. and Fukushima. S. Medium-term bioassay for carcinogens using DMA is used as a herbicide or pesticide in various areas of the world. multiorgan models. In: N. Ito and H. Sugano (eds.). Modification of Tumor Devel Moreover, many people are consistently exposed to As by environ opment in Rodents. Prog. Exp. Tumor Res., Vol. 33, pp. 41-57, Basel: Karger, 1991. mental or occupational routes (4-6, 8, 9). Now with the availability 20. Fukushima, S., Hagiwara, A., Hirose, M., Yamaguchi, S., Tiwawech, D., and Ito. N. Modifying effects of various chemicals on preneoplastic and neoplastic lesion devel of an animal model of As carcinogenesis, progress might be more opment in a wide-spectrum organ carcinogenesis model using F344 rats. Jpn. J. rapidly made in furthering our understanding of human risk to As Cancer Res., 82: 642-649, 1991. 21. Hirose. M.. Tanaka, H.. Takahashi, S.. Futakuchi. M.. Fukushima. S.. and Ito, N. exposures. Effects of sodium nitrite and catechol, 3-methoxycatechol, or butylated hydroxyani- sole in combination in a rat multiorgan carcinogenesis model. Cancer Res., 53: 32-37, 1993. ACKNOWLEDGMENTS 22. Babaya, K., Izumi, K., Ozono, S., Miyata, Y.. Morikawa, A., Chimel, J. S., and Oyasu, R. Capability of urinary components to enhance ornithine decarboxylase We are indebted to Dr. Manzo Nagahama, late Director of Osaka City activity and promote urotherial tumorigenicity. Cancer Res., 35: 2426-2433, 1975. 23. Matsui. Y. I., Otani, S., Yano, Y., Takada, N., Shibata, M. A., and Fukushima, S. Institute of Public Health and Environmental Sciences, for his assistance in this Spermidine/spermine W'-acctyltransferase, a new biochemical marker for epithelial experiment. We gratefully acknowledge the advice of Professor Samuel M. proliferation in rat bladder. Jpn. J. Cancer Res., 83: 1037-1040, 1992. Cohen at the University of Nebraska Medical Center in the preparation of this 24. Murai. T., Iwata, H.. Otoshi, T., Endo, G., Horiguchi. S., and Fukushima. S. Renal manuscript. We are grateful to Katsuhiko Kawabata and Yoshinori Inoue, lesions induced in F344/DuCrj rats by 4-weeks oral administration of dimethylarsinic Yokogawa Analytical System, Inc. (Tokyo, Japan) and Satoshi Uwagawa, acid. Toxicol. Lett., 66: 53-61, 1993. 25. Young, J. T. Histopathologic examination of the rat nasal cavity. Fundam. Appi. Sumitomo Chemical Co. Ltd. (Osaka, Japan) for measurement of As concen Toxicol., /.•309-312, 1981. trations by IC-ICP-MS and for assessment of prcneoplastic lesions with the aid 26. Ito, N.. Tsuda, H., Tatematsu. M., Inoue. T., Tagawa, Y., Aoki, T., Uwagawa, S., of a color video image processor (VIP-21C). We extend our gratitude to the Kagawa, M., Ogiso, T.. Masui. T., Imaida. K., Fukushima, S., and Asamoto, M. late Professor Kiyomi Sato of the Second Department of Biochemistry, Hiro- Enhancing effect of various hepatocarcinogens on induction of preneoplastic gluta- thione S-transferase placcntal form positive foci in rats—an approach for a new saki University, and Dr. Chic Furihata, Department of Molecular Oncology, medium-term bioassay system. Carcinogenesis (Lond.). 9: 387-394, 1988. Institute of Medical Sciences, University of Tokyo, respectively, for generous 27. Tatematsu. M., Furihata, C., Katsuyama, T., Mera, Y., Inoue, T., Matsushima, T., and provision of purified antibody against GST-P and anti-rat Pgl antibody. We Ito, N. Immunohistochcmical demonstration of pyloric gland-type cells with low- thank Dr. Ryohei Hasegawa, at the Department of Pathology, Nagoya City pepsinogen isozyme 1 in preneoplastic and neoplastic tissues of rat stomachs treated with W-methyl-W-nitro-W- nitrosoguanidine. J. Nail. Cancer Inst., 78: 771-777, 1987. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. Cancer Induction by an Organic Arsenic Compound, Dimethylarsinic Acid (Cacodylic Acid), in F344/DuCrj Rats after Pretreatment with Five Carcinogens
Shinji Yamamoto, Yoshitsugu Konishi, Tsutomu Matsuda, et al.
Cancer Res 1995;55:1271-1276.
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