Ozone Selectively Inhibits Growth of Human Cancer Cells Science Vol. 209, 22 Aug 1980, pp. 931-933 Abstract: The growth of human cancer cells from lung, breast, and uterine tumors was selectively inhibited in a dose-dependent manner by ozone at 0.3 to 0.8 part per million of ozone in ambient air during 8 days of culture. Human lung diploid fibroblasts served as noncancerous control cells. The presence of ozone at 0.3 to 0.5 part per million inhibited cancer cell growth 40 and 60 percent, respectively. The non-cancerous lung cells were unaffected at these levels. Exposure to ozone at 0.8 part per million inhibited cancer cell growth more than 90 percent and control cell growth less than 50 percent. Evidently, the mechanisms for defense against ozone damage are impaired in human cancer cells. The effects of ozone on human health have been a focus of public concern and scientific investigation for more than two decades (I-4). Considerable attention has been devoted to assessing its cellular effects (5) because it is the major constituent of the ground-level oxidants in polluted air. Much has been learned about the effects of ozone on normal tissue, but little is known about its action on cancer cells. We have conducted experiments in which continuous exposure to ozone at 0.3 ppm (6) selectively inhibited the growth of human cancer cells 40 percent in 8 days. Controlled levels of ozone (0.3 to 0.8 ppm) were continuously generated by ultraviolet irradiation of filtered ambient air. The ozonated air, containing 5 percent carbon dioxide, was introduced at a constant flow rate of 4.0 liter/min into an environmental chamber in an incubator maintained at 37 degrees Celsius (Fig. 1). The ozone levels were assayed daily with a spectrophotometric ozone analyzer. For comparison, noncancerous human lung diploid fibroblasts (7) were cultured in the chamber along with the cancer cells. The cancer cells were from alveolar (lung) adenocarcinomas (8), breast adenocarcinomas (9), uterine carcinosarcomas, and endometrial carcinomas (10). All the cells were grown in 60-mm petri dishes in 10 ml of medium and were placed in the chamber at the same time. Control cells were incubated in an adjoining compartment receiving filtered ambient air containing 5 percent carbon dioxide (4.0 liter/min). Three petri dishes for each cell type were removed from each of the two compartments every 48 hours for 8 days, and the number of cells per plate were counted. All of the cancer cells showed marked dose-dependent growth inhibition in ozone at 0.3 and 0.5 ppm (Fig 2.). There was no growth inhibition of the noncancerous lung cells at these ozone levels, and they were morphologically identical to the corresponding control cells. At 0.8 ppm, the growth of the noncancerous cells was inhibited 50 percent, but all four types of cancer cells were inhibited more than 90 percent. After being cultured through 14 passages, the noncancerous cells exhibited measurable growth inhibition and morphological changes (vacuolation) in ozone at 0.5 ppm, suggesting that aging increases the sensitivity of normal lung cells to ozone (Fig 3). In cultured human diploid fibroblasts, morphological changes and a gradual decrease in rate of growth have been attributed to a buildup of cellular damage with each successive division (11,12). Ozone may accelerate processes similar to those naturally causing cellular damage and may decrease the growth rate of the aging fibroblast colony. However, in ozone at 0.5 ppm, all of the human cancer cells (which do not age) had growth rates several times lower than that of the aged, noncancerous cells (Fig 2.). Evidently, cancer cells are less able to compensate for the oxidative burden of ozone than normal cells. The marked sensitivity of cancer cells to ozone raises questions about the possible mechanisms of oxidative inhibition of their growth. Virtually every major component of normal cells has been found to be affected by elevated ozone levels (5). However, glutathione in its reduced form (GSH) has been credited with providing the first line of defense against the peroxides and free radicals generated in all cells by ozone and oxygen (1, 13- 15). It deactivates peroxides and radicals by donating one hydrogen atom to the reactive species. Loss of a GSH hydrogen (oxidation) results in formation of oxidized glutathione (GS- SG). The cellular respiratory system is responsible for reducing GS-SG to GSH. The GSH-linked respiratory system in normal and cancer cells, before and after exposure to ozone, must be examined to learn whether a functional impairment of this system is associated with the marked sensitivity of cancer cells to the oxidant. These findings lead us to believe that ozone--alone, in combination with radiation therapy (16), or in chemotherapy utilizing electrophilic compounds (17)--may have therapeutic value for patients with certain forms of lung cancer. Frederick Sweet Ming-Shian Kao Song-Chiau D. Lee Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri, 633110. Will L. Hagar City of St. Louis Air Pollution Control, St. Louis, 63103 Wileen E. Sweet Air Quality Section, East-West Gateway Coordinating Council, St. Louis, 63102. Figure 1. Schematic diagram (not shown) of the system used for culturing human cells in ozonated ambient air. Filtered ambient air was mixed with carbon dioxide (5 percent) and introduced into a dual chamber incubator (National 331). Half was conducted through a calibrated ozone generator consisting of a quartz glass tube irradiated with ultraviolet light and then into a hermetically sealed (20 by 20 by 20 cm) glass and stainless steel environmental chamber containing a gasketed access door. Output of ozone from the generator varied less than 1 percent per day. The ozone content of the vented air from the chamber was measured daily with a spectrophotometric ozone analyzer (Dasibi 1003-AH). Malignant and normal human cells were incubated in chamber E saturated with water vapor. Corresponding cells serving as controls were incubated in the adjoining compartment, also saturated with water vapor. Figure 2. Inhibition by ozone of growth of malignant and non- malignant cells in culture on day 8. Each of the cell types were grown in 10 ml of Dulbecco's modified Eagle's minimum essential medium containing 10 percent calf serum. In a typical experiment, 12 dishes per cell line (usually three or four cell lines were tested per experiment) were loaded into the environmental chamber with an equal number of control dishes in the adjoining compartment (Fig. 1). The initial population was 3 x 10(5) cells per dish. Every 48 hours three dishes for each cell type were removed from both compartments and the cells were tested for viability with 0.4 percent trypan blue and counted with a hemocytometer. Each data point represents the number of experimental cells divided by the number of corresponding control cells per dish multiplied by 100 (the percentage of control growth) and is plotted against the measured level of ozone in the environmental chamber. The percentage of growth inhibition is calculated by subtracting the percentage of growth from 100. The data are from cell counting on day 8 of incubation. There is a nearly linear relation between inhibition of the growth of the cancer cells and increasing ozone levels. The noncancerous cell line IMR-90 began to display measurable growth inhibition only when ozone levels exceeded 0.5 ppm, a level that produced approximately 60 percent inhibition in all of the cancer cells lines tested. There was some growth inhibition in noncancerous cells aged through 14 passages. The mean populations of the cells serving as controls were as follows (per dish on day 8): IMR-90, 34.8 x 10(5); A- 549, 36.5 x 10(5); MCF-7, 57.0 x 10(5); endometrial adenocarcinoma, 64.2 x 10(5); myometrial carcinosarcoma, 121.1 x 10(5). References: 1. D.L. Dunsworth, C.E. Cross, J.R. Gillespie, C.G. Plopper, in Ozone Chemistry and Technology. J.S. Murphy and J.R. Orr, Eds. (Franklin Institute, Philadelphia, 1975), chap. 2. 2. H.E. Stokinger and D. Coffin, in Air Pollution, A.C. Stern, Ed. (Academic Press, New York, 1968), vol. 1, pp. 446-546. 3. H.D. Kerr et al., Am. Rev. Respir. Dis. 111, 763 (1975). 4. J.D. Hackney, W.S. Linn, C.D. Law, S.K. Karuza, Greenberg, R.D. Buckley, E.E. Pedersen, Arch. Environ. Health 30, 385 (1975). 5. B.D. Goldstein, Rev. Environ. Health 2, 177 (1977). 6. Normal human subjects tolerated breathing 0.5 ppm ozone in air 2 hours per day for 1 week or 0.25 ppm ozone 2 hours per day for 3 weeks (4). The two groups engaged in light exercise during exposure. Although both groups developed chest discomfort and moderately decreased respiratory function during exposure, their removal from the oxidative environment resulted in rapid disappearance of the symptoms. The mean dose-response curves from this study show a no- detectable-effect level at 0.25 to 0.30 ppm. A similar study (3) found that human subjects tolerated exposure to 0.5 ppm ozone for up to 6 hours. Pulmonary function was affected and chest discomfort developed at this level, with no significant differences observed between smokers and nonsmokers. 7. These cells (IMR-90) were obtained from the Human Aging Cell Repository and plated 48 hours after shipping. This cell type was characterized by W.W. Nichols, D.G. Murphy, V.I. Cristofalo, L.H. Toji, A.E. Greene, and S.A. Dwight [Science 196, 60 (1977)]. 8. This cell line (A-549) was described by D.J. Glard, S.A. Aaronson, G.J. Todard, P. Arnstein, J.H. Kersey, H. Dorsik, and W.P. Parks [J. Natl. Cancer Inst. 51, 1417 (1973)]; M. Lieber, B. Smith, A. Szakal, W. Nelson-Rees, and G.A. Todardo [Int. J. Cancer 17, 62 (1976)]; and K.L. Jones, N.S. Anderson III, and J. Addison [Cancer Res. 38, 1688 (1978)] 9. This cell line (MCF-7, estrogen-sensitive) was described by H.D. Soule, J. Vazquez, A. Long, S. Albert, and M. Brennam [J. Natl. Cancer Inst. 51, 1409 (1973)] and by K.B. Horwitz, M.E. Kostlow, and W.I. McGuire [Steroids 26, 785(1975)]. 10. Human uterine carcinosarcoma cells and endometrial adenocarcinoma cells were obtained from pathologically confirmed gynecologic tumors and developed as new cell lines. The endometrial adenocarcinoma cell line is estrogen-sensitive. Both were described by M.S. Kao and S.C.D. Lee (27th Annual Meeting of the Society for Gynecologic Investigation, Denver, 20 to 23 March 1980), abstr. 7. 11. J.R. Smith and R.G. Whitney, Science 207, 82 (1980); S.C.D. Lee, P.M. Bemiller, J.N. Bemiller, A.J. Papelis, Mech. Ageing Dev. 7, 417 (1978). 12. P.M. Bemiller and L.H. Lee, ibid. 8, 417 (1978) 13. C.K. Chow and A.L. Tappel, Lipids 1, 518 (1972). 14. C.K. Chow, Nature (London) 260, 721 (1976). 15. R.E. Kimball et al., Am. J. Physiol. 230, 1425 (1976). 16. R.E. Lee, Semin. Oncol. 1, 254 (1974) 17. O.S. Selawry, ibid., p. 259. 18. Parts of this report were presented at the 27th Annual Meeting of the Society for Gynecologic Investigation, Denver, 20 to 23 March 1980 (abstracts 7 and 150), and at the 73rd Annual Meeting of the Air Pollution Control Association, Montreal, 24 June 1980 (poster session 27). We thank W. Nelson-Rees for his gift of A-549 cells; the MCF-7 cells were obtained from E.M. Jensen. We also thank C.M. Copley, Jr., H.M. Camel, and T. Morgan for their constructive criticism of the manuscript. Correspondence should be addressed to F.S. 24 April 1980; revised 11 June 1980.