Preliminary experimental study on carcinogenicity of arsenic trioxide in rat lung.

To investigate carcinogenic or cocarcinogenic properties, copper ore (Kinkaseki) and flue dust collected from a metal refinery and arsenic trioxide (As2O3) were administered into the lung of male Wistar-King rats by an intratracheal instillation method. No squamous cell carcinoma of the lung was found among the rats given three arsenical substances, while adenoma or adenocarcinoma was observed. Squamous cell carcinoma of the lung was observed in rats, when copper ore, flue dust, and arsenic trioxide were instilled into the lung together with benzo[a]pyrene (B[a]P. The incidence of squamous cell carcinoma of the lung in rats exposed to Kinkaseki, flue dust, and As2O3 in addition to B[a]P was higher than that in rats given B[a]P alone. The results of this study indicate that solid arsenical substances, such as arsenic trioxide, metal ore and flue dust from a metal refinery, seemed to act on the carcinogenicity of B[a]P in a cocarcinogenic manner. ImagesFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 4.FIGURE 5.FIGURE 6.


Introduction
Inorganic arsenical compounds, including arsenic trioxide, have been suspected as human carcinogens in several epidemiological studies on workers in mines and copper smelters (1)(2)(3). Many clinical studies, epidemiological, occupational and therapeutic exposure studies have shown the same trend. Recently, Tokudome and Kuratsune (4) reported that the risk of lung cancer was significantly increased in copper smelter workers in a metal refinery in Japan. In spite of the epidemiological events mentioned above, there are essentially negative reports concerning malignant lung tumors in animals following exposure to inorganic arsenical compounds given through the airway into the lung. In the present study, arsenic trioxide, Kinkaseki (copper ore), and flue dust containing some amounts of arsenic collected from the metal refinery reported by Tokudome and Kuratsune (4) were examined for carcinogenicity and cocarcinogenicity with benzo[a]pyrene (B[a]P) in rats. *Department of Hygiene, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
These materials were intratracheally instilled into the lung of rats.

Materials and Methods
Kinkaseki ore and the flue dust were collected from a metal refinery, Oita Prefecture, Japan, in May, 1971. These materials were transferred into a Soxhlet apparatus and were extracted with acetone to remove tarry materials which might contain carcinogenic hydrocarbons. After repeated extraction, it could be ascertained that these materials did not contain B[a]P and did not have any fluoresence.
Metals in Kinkaseki and the flue dust were qualitatively determined by emission spectroanalysis; the results obtained are shown in Table 1. Arsenic, nickel,-chromium, copper, iron, and manganese were analyzed quantitatively by flameless or flame. atomic absorption spectroanalysis, and the results are shown in Table 2. Arsenic concentrations were 3.95% in Kinkaseki and 10.6% in the flue dust.
Arsenic trioxide, analytical grade, from Katayama-Seiyaku Co., Japan, was used in the experiment.
Benzo[a]pyrene used in the experiment was manufactured by Sigma Ltd., Basel, Switzerland.  Saline (supplied by the Hospital Dispensary, Kyushu University) was used for the control group in the experiment.
Male Wistar-King albino rats, about 10 weeks old, came from the colony of the Animal Center, Kyushu University. The rats were divided into eight groups according to the following exposure regimen (shown in Table 3 group. An intratracheal instillation method was used (5). The rats were given an injection of atropine sulfate subcutaneously and were anesthetized with a mixture of 5% ether and 95% oxygen in a desiccator for 5 min. The anesthetized rat was removed from the desiccator and 0.2 ml of the suspended materials in distilled water or saline was instilled into the lung by a microsyringe with a special metal needle. The needle was carefully inserted into the distal part of the trachea by viewing the slit of the pseudo-vocal cords through magnifying glasses.
Each suspension was prepared as follows. About 20 ml of each suspension was neutralized with dilute sodium hydroxide solution, and the solutions were homogenized, sterilized, and suspended for 10 min with an ultrasonic wave generator (Taisho Denki Ltd., Japan) under nitrogen gas. The distributions of particle sizes, of Kinkaseki, flue dust, and As203 in the suspensions were as follows; 0.45% -10 ,u, 2.00% 10-5 ,u, 97.55% < 5 ,u for Kinkaseki; 2.72%, 5.35%, and 91.93%, respectively, for flue dust; 1.06%, 1.27%, and 97.67% respectively, for AS203. The particle size of B[a]P in the suspension was about 5 , or less. In all suspensions, especially in flue dust, considerable aggregation of particles were observed. All rats received 15 exposures in all, once a week for about 4 months. The number of surviving rats among experimental groups after the 15th instillation were as follows; 10 in the-group receiving Kinkaseki, 7 in the flue dust group, 8 in As203 group, 7 in the B[a]P group, 10 in the Kinkaseki + B[a]P group, 10 in the flue dust + B[a]P group, 7 in the As203 + B[a]P group, and 7 in the saline group, as shown in Table 3. They were raised on commercial solid foods (Oriental NMF, Orient-al Co. Japan) and were given drinking water ad 14ium. All surviving rats were observed during their entire life span and allowed to die spontaneously. The animals were autopsied, and main visceral organs and any tumors of tissues or organs were fixed with a 10% formalin/water solution. The respiratory organs were especially carefully examined with magnifying glasses. The lung was inflated and fixed by filling 10%o formalin/water solution with a syringe through trachea. For microscopical histopathological examination, sections (about 6 u) were prepared and stained with hematoxylin-eosin. For precise examination, sections were prepared from two to six blocks from each of the five lobules of the lung. Environmental Health Perspectives Results observed in I of 10 survivors in the Kinkaseki group and I out of 8 survivors in the As203 group. In the As shown in Table 3, in the eight experimental flue dust group, adenocarcinoma was observed in I groups, the average survival after 15 instillations out of 7 survivors. In histopathological examinaranged from 372 days in the (copper ore + B[a]P) tion, all rats of the above mentioned three groups group to 837 days in the B[a]P group. In three had squamous cell metaplasia in the airway or osgroups, Kinkaseki (copper ore), flue dust, and teometaplasia in the alveolus of the lung as shown As203, induction of squamous cell carcinoma was in   (Figs. 3-5). Furthermore, squamous cell metaplasia in the lining cells of the airway and osteometaplasia in the alveolar cells of the lung were observed in almost all rats as shown in Table 4 (Fig. 6). No incidence of malignant or benign tumors existed in rats of the saline group, and other histopathological changes in these rats were much less than those of other groups. There is squamous cell carcinoma with pearl formation in the right lower lobule of the lung. H. E. stain x 59.
The incidence rates of malignant lung tumors of rats in the groups; the combined group of rats which received the different arsenical substances, the B[a]P group, the combined group of rats receiving arsenical substances plus B[a]P, and the saline group, were statistically compared with each other. There was a significant difference of incidence rate only between the combined group of three arsenical substances (1 out of 25 rats, 4.0%) and the combined group of three arsenical substances plus B [a]P (8 out of 27 rats, 29.6%) as shown in Table 3.
The tumor incidence rate in each group (malignant tumors plus benign tumors) is shown in Table 4, and the tumor incidence rate of the lung in the combined group of three arsenical substances was 12%, that is 3 out of 25 rats.  stain, x 66.

Discussion
The number of rats surviving in each group afte1 15 instillations in this experiment was not adequate to confirm carcinogenicity of arsenic to the lung. Squamous cell carcinoma of the lung in Wistar-King rats could not be induced by intratracheal instillation of arsenical substances. Spontaneous lung tumors are rare in Wistar-King rats; however, adenoma and adenocarcinoma of the lung were ob-served in the rats among three groups exposed intratracheally to arsenical substances. Taking account of the tumorigenesis mentioned above, carcinogenicity of arsenic itself to the lung of animal cannot be absolutely denied in the field of animal experiment.
The results of this study indicate that solid arsenical substances such as arsenical trioxide, metal ore, and flue dust, containing some amount of arsenic, from a metal refinery seemed to act on the carcinogenicity of B[a]P in a cocarcinogenic manner. The incidence of squamous cell carcinoma of the lung in rats exposed to Kinkaseki (copper ore), flue dust, and As203 plus B[a]P was higher than that of B[a]P alone. The cocarcinogenic action of other solid substances as well as arsenicals to inducing lung tumor in animals were reported by Saffiotti et al. (6) and Feron (7). In epidemiological consideration of high risk of lung cancer among workers in copper smelters, Lee and Fraumeni (1) and also Tokudome and Kuratsune (4) suggested an association for carcinogenicity between arsenic trioxide and other carcinogens or/and cocarcinogens, such as polycyclic aromatic hydrocarbons and sulfur dioxide. In conclusion, this experiment indicates that carcinogenicity of inorganic arsenical compounds, particularly arsenic trioxide, for aniYnals, cannot be absolutely ruled out by the method of intratracheal administration. Further animal experiments by intratracheal instillation technique on larger numbers of animals and with higher dosages of inorganic arsenicals are necessary.

Summary
Carcinogenicity to the lung of Kinkaseki (copper ore), the flue dust collected from a metal refinery in Japan, and arsenic trioxide (As203) was studied in male Wistar-King rats by an intratracheal instillation method. Simultaneously carcinogenicity to the lung of B[a]P, Kinkaseki plus B[a]P, flue dust plus B[a]P, As203 plus B[a]P also was examined in the rats by the same method. In the experiment, 2.5 mg of Kinkaseki, 2.0 mg of flue dust, and 0.26 mg of As203 were instilled intratracheally into the lung once a week for 15 weeks. No squamous cell lung cancer was observed. One adenoma out of 10 rats in the Kinkaseki group, one adenocarcinoma out of 7 rats in the flue dust group, and one adenoma out of 8 rats in the As203 group were observed.
When the same method of instillation was used on other groups of rats, 0.