Evaluation of Oxidative Stress Parameters and Genotoxic Effects in Patients With Work-Related Asthma and Silicosis.

OBJECTIVE: To determine the mechanistic roles of oxidative stress, inflammation, and genotoxicity parameters in patients with work-related asthma (WRA) and silicosis. METHODS: Thirty-eight healthy office workers, 27 employees with a history of exposure and no disease, 24 employees with WRA, and 23 employees with silicosis were included in this study. Superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, and interleukins (IL) 17, 23, and 27 levels were measured in the serum. Genotoxic damage was evaluated by calculating the frequency of micronuclei in swab samples and 8-hydroxy-2'-deoxyguanosine in serum. RESULTS: Serum superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and IL-17, IL-23, and IL-27 levels were found to be statistically significantly higher in the exposure, WRA, and silicosis groups compared with the control group. The frequency of micronuclei in buccal epithelial cells of the patient group was found to be significantly higher than that of the control group. CONCLUSION: These results may provide information for molecular mechanisms and early diagnosis of WRA and silicosis and will be a guide for taking precautions in the early period.

Protective Effects of Antioxidant Chlorophyllin in Chemically Induced Breast Cancer Model In vivo.

Glutathione-related enzymes belong to the protection mechanism of the cells against harmful oxidative damage and chemicals. Glutathione S-transferase (GST) is frequently over-expressed in various cancer cells and is involved in drug resistance. Chlorophyllin is an antioxidant molecule interfering with the GST P1-1 activity. The purpose of this study is to evaluate the short- and long-term protective effects of chlorophyllin as an antioxidant molecule on DNA damage, antioxidant enzyme activities, trace elements, and minerals in chemically induced breast cancer model in vivo. In our study, N-methyl-N-nitrosourea (MNU) was used for inducing breast carcinogenesis in female Sprague-Dawley rats. A total of 36 rats were divided into groups as short term and long term. Each group was divided into four sub-groups as control group received physiological saline solution (n = 3), Chl group (n = 5) received chlorophyllin, MNU group (n = 5) was administered MNU, and Chl + MNU group (n = 5) was treated with both chlorophyllin and MNU. Results illustrated that chlorophyllin had a significant anti-genotoxic effect in the short term, and glutathione-related enzyme activities were protected by chlorophyllin treatment in MNU-induced breast cancer model. Additionally, MNU administration impaired mineral and trace element levels including Na, Mg, K, Fe, Zn, and Co in the liver, kidney, spleen, heart, and tumor tissues; however, adverse effects of MNU were recovered upon chlorophyllin treatment in the indicated tissues of the rats. In conclusion, chlorophyllin can be used as an antioxidant molecule to ameliorate adverse effects of MNU by enhancing antioxidant enzyme activities and regulating trace element and mineral balance in several organs and tumor tissue in the breast cancer model.

Evaluation of the Possible Role of miRNAs in Chemical Allergen Potency.

OBJECTIVES: MicroRNAs (miRNAs) are short, endogenous noncoding RNA molecules that can bind to certain parts of target mRNAs, thereby regulating gene expression. Studies showed that miRNAs could be up- or downregulated in different allergic skin conditions but there is still need for further studies. The aim of this study was to investigate the expression of miRNAs in response to the common contact allergen Bandrowski's base (BB), the principal allergen in patients reacting to p-phenylenediamine (PPD). MATERIALS AND METHODS: The human promyelocytic cell line THP-1 was exposed to BB at a concentration of 1 µg/mL for 24, 48, and 72 h. The dose was selected from the results of cytotoxicity assays. RNA was purified and miRNA expression profile and real-time polymerase chain reaction (RTPCR) were performed to identify up- or downregulated miRNAs and confirm their modulations. RESULTS: Among the different modulated miRNAs, the upregulation of miRNA-155 and the downregulation of miRNA-21 were found to be important because these are related to immune system. This expression profile of miRNAs was also confirmed by RT-PCR. CONCLUSION: These preliminary results showed that miR-155 and miR-21 may play a role in the pathogenesis of allergic contact dermatitis, but further studies are needed to clarify their definite roles.

Oxidative stress status of Turkish welders.

Welding, a fabrication process that joins metals or thermoplastics by causing coalescence, is indispensable in modern society and ubiquitous in industry. Welding generates fumes that contain several metals and gases that comprise fine and ultrafine particles with the potential for adverse effects. Although health risks of welders have been evaluated in different populations, occupational exposure to welding fumes is still considered to be an important health problem, especially in developing countries. The aim of this study was to investigate the effects of welding fume exposure on important oxidative stress parameters such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), total glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in Turkish welders (n = 48). The influence of confounding factors such as age, smoking habits, alcohol consumption, and duration of exposure on the studied parameters was also analyzed. In our study, significant decreases in the levels of GSH and activities of CAT, SOD, and GPx and significant increases of MDA, 8-OHdG levels and GR activity were found in the workers compared to the controls. There was a negative correlation between GSH levels and alcohol usage. Also, older workers (≥35 years) had significantly higher GR levels than younger workers. But smoking and alcohol usage, duration of exposure, and utilization of protective measures had no significant effect on the studied parameters in the workers. These results indicate that occupational exposure to welding fumes appears to induce oxidative stress and oxidative DNA damage.

Assessment of DNA damage in welders using comet and micronucleus assays.

Welding technology is widely used in pressurized containers, thermal power plants, refineries, chemical facilities and steel structures. Welders are exposed to a number of hazardous compounds such as ultraviolet (UV) radiation, electromagnetic fields, toxic metals and polycyclic aromatic hydrocarbons (PAHs). In the present study, 48 welders and an equal number of control subjects were evaluated for DNA damage in the whole blood and isolated lymphocytes using the comet assay. The genotoxic damage in buccal epithelial cells of subjects was determined by micronucleus (MN) assay. Metal(loids) such as Cr, Mn, Ni, Cu, As, Cd and Pb levels in blood samples were evaluated by using Inductively Coupled Plasma-Mass Spectrometer (ICP-MS). Results of this study showed that DNA damage in blood, isolated lymphocytes, and buccal epithelial cells were significantly higher in workers compared to the controls. Also, these workers had remarkably higher blood Cr, Cu, Cd, Ni and Pb levels. These results showed that occupational exposure to welding fumes may cause genotoxic damage that can lead to important health problems in the workers. More extensive epidemiological studies should be performed that enable the assessment of health risk in welding industry.

Evaluation of the DNA damage in lymphocytes, sperm and buccal cells of workers under environmental and occupational boron exposure conditions.

Industrial production and use of boron compounds have increased during the last decades, especially for the manufacture of borosilicate glass, fiberglass, metal alloys and flame retardants. This study was conducted in two districts of Balikesir; Bandirma and Bigadic, which geographically belong to the Marmara Region of Turkey. Bandirma is the production and exportation zone for the produced boric acid and some borates and Bigadic has the largest B deposits in Turkey. 102 male workers who were occupationally exposed to boron from Bandirma and 110 workers who were occupationally and environmentally exposed to boron from Bigadic participated to our study. In this study the DNA damage in the sperm, blood and buccal cells of 212 males was evaluated by comet and micronucleus assays. No significant increase in the DNA damage in blood, sperm and buccal cells was observed in the residents exposed to boron both occupationally and environmentally (p = 0.861) for Comet test in the sperm samples, p = 0.116 for Comet test in the lymphocyte samples, p = 0.042 for micronucleus (MN) test, p = 0.955 for binucleated cells (BN), p = 1.486 for condensed chromatin (CC), p = 0.455 for karyorrhectic cells (KHC), p = 0.541 for karyolitic cells (KLY), p = 1.057 for pyknotic cells (PHC), p = 0.331 for nuclear bud (NBUD)). No correlations were seen between blood boron levels and tail intensity values of the sperm samples, lymphocyte samples, frequencies of MN, BN, KHC, KYL, PHC and NBUD. The results of this study came to the same conclusions of the previous studies that boron does not induce DNA damage even under extreme exposure conditions.

Environmental boron exposure does not induce DNA damage in lymphocytes and buccal cells of females: DNA damage in lymphocytes and buccal cells of boron exposed females.

Boron (B) compounds are essential for plants and animals and beneficial for humans in nutritional amounts. I animals and humans increasing evidence have shown beneficial effects on B compounds on nutrition and on antioxidant status. The genotoxic effects of environmental B exposure in women living in boron-rich and boron-poor areas was examined in this study. For this purpose, the DNA damage in the lymphocytes and buccal cells of females were assessed by Comet and micronucleus (MN) assays respectively. No significant difference was observed in the DNA damage of the lymphocytes of B exposed groups of female volunteers in Comet assay. Even buccal micronucleus (MN) frequency observed in the high exposure group was significantly lower than the low exposure group (p < 0.05). The results of this study came to the same conclusions of the previous studies that boron does not induce DNA damage even under extreme exposure conditions.

Boron-exposed male workers in Turkey: no change in sperm Y:X chromosome ratio and in offspring's sex ratio.

Boron-associated shifts in sex ratios at birth were suggested earlier and attributed to a decrease in Y- vs. X-bearing sperm cells. As the matter is pivotal in the discussion of reproductive toxicity of boron/borates, re-investigation in a highly borate-exposed population was required. In the present study, 304 male workers in Bandirma and Bigadic (Turkey) with different degrees of occupational and environmental exposure to boron were investigated. Boron was quantified in blood, urine and semen, and the persons were allocated to exposure groups along B blood levels. In the highest ("extreme") exposure group (n = 69), calculated mean daily boron exposures, semen boron and blood boron concentrations were 44.91 ± 18.32 mg B/day, 1643.23 ± 965.44 ng B/g semen and 553.83 ± 149.52 ng B/g blood, respectively. Overall, an association between boron exposure and Y:X sperm ratios in semen was not statistically significant (p > 0.05). Also, the mean Y:X sperm ratios in semen samples of workers allocated to the different exposure groups were statistically not different in pairwise comparisons (p > 0.05). Additionally, a boron-associated shift in sex ratio at birth towards female offspring was not visible. In essence, the present results do not support an association between boron exposure and decreased Y:X sperm ratio in males, even under extreme boron exposure conditions.

DNA damage assessment with buccal micronucleus cytome assay in Turkish coal miners.

The aim of this study was to assess DNA damage in Turkish coal miners with the buccal micronucleus cytome (BMCyt assay as the least invasive and therefore most practical method that may find wider application in coal miner biomonitoring. Buccal epithelial cell samples were taken from 54 coal miners and 42 controls from Zonguldak, Turkey to establish their micronucleus (MN), binucleus (BN), condensed chromatin (CC), karyorrhectic (KHC), karyolytic (KYL), nuclear bud (NBUD), and pyknotic (PYC) frequencies. We also analysed the effects of confounding factors such as age, years of work at the mine, smoking, alcohol drinking, and use of protective equipment on differences in MN frequencies. Two miners had confirmed and three suspect pneumoconiosis, whereas 49 displayed normal chest radiographs. MN, BN, KHC, and NBUD frequencies were significantly higher in coal miners than controls. Years of work at the mine also showed a significant effect on buccal MN frequencies in coal miners, but we found no correlation between MN frequencies and age, smoking, and alcohol consumption. In conclusion, BMCyt assay proved itself an accurate and practical screening method, as it can detect DNA damage much earlier than pneumoconiosis develops.

Dual actions of the antioxidant chlorophyllin, a glutathione transferase P1-1 inhibitor, in tumorigenesis and tumor progression.

Glutathione (GSH) and enzymes related to this antioxidant molecule are often overexpressed in tumor cells and may contribute to drug resistance. Blockade of glutathione transferases (GSTs) has been proposed to potentiate the efficacy of chemotherapeutic drugs in cancer. The aim of this study was to evaluate the effect of chlorophyllin that has antioxidant properties, and also interferes with the activity of GST P1-1, on breast cancers in vitro and in vivo. The in vivo studies were conducted using an N-methyl- N-nitrosourea (MNU)-induced chemical carcinogenesis model in laboratory rats. DNA damage, GST activity, and GSH levels were determined in liver and tumor tissues. Treatment with chlorophyllin increased the GSH levels in the liver and significantly decreased DNA damage in the blood, liver, and tumor tissues. Even though tumorigenesis was delayed in rats receiving chlorophyllin before MNU injections, once the tumors emerged, the progression of tumor appeared to be faster than in the animals that received the carcinogen only. Out of nine breast cell lines, GST P1-1 expression was detected in MCF-12A, MDA-MB-231, and HCC38. Concomitant incubation with chlorophyllin and docetaxel did not significantly affect cell proliferation and viability. Chlorophyllin displayed genoprotective effects that initially delayed tumorigenesis. However, once the tumors were established, it may act as a promoter that facilitates tumor growth, potentially by a mechanism independent of cell proliferation and viability. Our results underline the pros and cons of antioxidant treatment in cancer, even if it has a capacity to inhibit GST P1-1.

Preventive role of Pycnogenol® against the hyperglycemia-induced oxidative stress and DNA damage in diabetic rats.

Diabetes mellitus, a complex progressive metabolic disorder, leads to some oxidative stress related complications. Pycnogenol® (PYC), a plant extract obtained from Pinus pinaster, has been suggested to be effective in many diseases including diabetes, cancer, inflammatory and immune system disorders. The mechanisms underlying the effects of PYC in diabetes need to be elucidated. The aim of this study was to determine the effects of PYC treatment (50 mg/kg/day, orally, for 28 days) on the DNA damage and biochemical changes in the blood, liver, and kidney tissues of experimental diabetic rats. Changes in the activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase enzymes, and the levels of 8-hydroxy-2'-deoxyguanosine, total glutathione, malondialdehyde, insulin, total bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, high density lipoprotein, low density lipoprotein, total cholesterol, and triglyceride were evaluated. DNA damage was also determined in the whole blood cells and the liver and renal tissue cells using the alkaline comet assay. PYC treatment significantly ameliorated the oxidative stress, lipid profile, and liver function parameters as well as DNA damage in the hyperglycemic rats. The results show that PYC treatment might improve the hyperglycemia-induced biochemical and physiological changes in diabetes.

Evaluation of FSH, LH, testosterone levels and semen parameters in male boron workers under extreme exposure conditions.

Boric acid and sodium borates are currently classified in the EU-CLP regulation as "toxic to reproduction" under "Category 1B", with hazard statement of H360FD. However, so far field studies on male reproduction in China and in Turkey could not confirm such boron-associated toxic effects. As validation by another independent study is still required, the present study has investigated possible boron-associated effects on male reproduction in workers (n = 212) under different boron exposure conditions. The mean daily boron exposure (DBE) and blood boron concentration of workers in the extreme exposure group (n = 98) were 47.17 ± 17.47 (7.95-106.8) mg B/day and 570.6 ± 160.1 (402.6-1100) ng B/g blood, respectively. Nevertheless, boron-associated adverse effects on semen parameters, as well as on FSH, LH and total testosterone levels were not seen, even within the extreme exposure group. With this study, a total body of evidence has accumulated that allows to conclude that male reproductive effects are not relevant to humans, under any feasible and realistic conditions of exposure to inorganic boron compounds.

Birth weights of newborns and pregnancy outcomes of environmentally boron-exposed females in Turkey.

Boric acid and sodium borates are currently classified as being toxic to reproduction under "Category 1B" with the hazard statement of "H360 FD" in the European CLP regulation. This has prompted studies on boron-mediated reprotoxic effects in male workers in boron mining areas and boric acid production plants. By contrast, studies on boron-mediated developmental effects in females are scarce. The present study was designed to fill this gap. Hundred and ninety nine females residing in Bandirma and Bigadic participated in this study investigating pregnancy outcomes. The participants constituted a study group covering blood boron from low (< 100 ng B/g blood, n = 143) to high (> 150 ng B/g blood, n = 27) concentrations. The mean blood boron concentration and the mean estimated daily boron exposure of the high exposure group was 274.58 (151.81-975.66) ng B/g blood and 24.67 (10.47-57.86) mg B/day, respectively. In spite of the high level of daily boron exposure, boron-mediated adverse effects on induced abortion, spontaneous abortion (miscarriage), stillbirth, infant death, neonatal death, early neonatal death, preterm birth, congenital anomalies, sex ratio and birth weight of newborns were not observed.

Effects of cinnamic acid on complications of diabetes

Background/aim: Diabetes mellitus (DM) is a major health problem worldwide. Cinnamic acid (CA) and its derivatives are synthesized in plants and increasing attention has been given to them in recent years due to the high number of beneficial health properties attributed to their consumption. The aim of this study was to investigate the effects of CA on streptozotocin-induced diabetes in Wistar albino rats. Materials and methods: DNA damage was evaluated in the blood, liver, and kidney cells of rats by the alkaline comet assay. Oxidative stress parameters such as catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase activities and 8-hydroxy-2-deoxyguanosine, total glutathione, and malondialdehyde levels; biochemical parameters including insulin, total bilirubin, and BCA protein levels; hepatic enzyme levels such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase; and lipid profile parameters including high-density lipoprotein, low-density lipoprotein, total cholesterol, and triglyceride levels were also evaluated. Results: DM caused genotoxic damage and alterations in lipid profiles, oxidative stress parameters, and hepatic enzymes levels. CA treatment ameliorated these effects. Conclusion: It seems that CA might have a role in the prevention of the complications of diabetes.

Protective Effects of Ursolic Acid in the Kidneys of Diabetic Rats.

OBJECTIVES: Diabetes, a heteregenous metabolic and chronic disease, is a growing health problem in most countries. It has been claimed that diabetes is associated with the increased formation of free radicals and decreased in antioxidant potential. Oxidative stress formed in diabetes may cause DNA damage in the tissues. Ursolic acid, a well-known pentacylic triterpene, is commonly used in traditional Chinese medicine due to its beneficial health effects such as antioxidant, anticancer, and antiulcer properties. The aim of this study was to investigate the effects of ursolic acid in the kidneys of Wistar albino rats with streptozotocin-induced diabetes. MATERIALS AND METHODS: DNA damage was evaluated in the kidney cells of rats using alkaline comet assays. Oxidative stress parameters such as CAT, SOD, GR, and GSH-Px enzyme activities and total GSH and MDA levels were also evaluated. RESULTS: Ursolic acid treatment was found to significantly decrease DNA damage, GR enzyme activities, and MDA levels, and significantly increase GSH levels and CAT, SOD and GSH-Px enzyme activities in diabetic rats. CONCLUSION: According to our results, it seems that ursolic acid may be beneficial against diabetes-induced renal damage.

Assessment of DNA damage in ceramic workers.

It is known that ceramic workers are potentially exposed to complex mixture of chemicals such as silica, inorganic lead, lime, beryllium and aluminum that can be associated with an increased risk of several diseases. All operations in the ceramic industries such as mixing, moulding, casting, shaking out and finishing jobs, have been associated with the higher exposure levels and in most of the silica-related industries, average overall exposure exceeded permissible exposure levels for respirable crystalline silica. The aim of this study was to evaluate the possible genotoxic damage in ceramic workers exposed to complex mixture of chemicals mainly crystalline silica. For this purpose, the blood and buccal epithelial cell samples were taken from the ceramic workers (n = 99) and their controls (n = 81). The genotoxicity was assessed by the alkaline comet assay in isolated lymphocytes and whole blood. Micronucleus (MN), binucleated (BN), pyknotic (PYC), condensed chromatin (CC), karyolytic (KYL), karyorrhectic (KHC) and nuclear bud (NBUD) frequencies in buccal epithelial cells and plasma 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels were also evaluated. In the study, 38 workers were diagnosed with silicosis, 9 workers were suspected to have silicosis, whereas 52 workers were found to be healthy. DNA damage in blood and lymphocytes; MN, CC + KHC, PYC frequencies in buccal epithelial cells and 8-oxodG levels in plasma were increased in workers compared to their controls. These results showed that occupational chemical mixture exposure in ceramic industry may cause genotoxic damage that can lead to important health problems in the workers.

d-limonene ameliorates diabetes and its complications in streptozotocin-induced diabetic rats.

It is known that diabetes causes some complications including alterations in lipid profile, hepatic enzyme levels but also it causes oxidative stress. Limonene, a major component of Citrus oils, has important health beneficial effects in lowering the level of oxidative stress due to its antioxidant activity. The aim of this study was to investigate the effects of D-limonene on streptozotocin (STZ)-induced diabetes in Wistar albino rats. For this purpose, DNA damage was evaluated by alkaline comet assay. Changes in the activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GSHPx) and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), total glutathione (GSH), malondialdehyde (MDA), insulin, total bilirubin and BCA protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol and triglyceride were also evaluated. D-limonene treatment was found to significantly decrease DNA damage, GR enzyme activities and MDA levels and significantly increase GSH levels and CAT, SOD and GSH-Px enzyme activities and altered lipid and liver enzyme parameters in diabetic rats. According to our results, it seems that D-limonene might have a role in the prevention of the complication of diabetes in rats.

Effects of Occupational Silica Exposure on OXIDATIVE Stress and Immune System Parameters in Ceramic Workers in TURKEY.

Silica is the second most common element after oxygen, and therefore, exposures to crystalline silica dust occur in a large variety of occupations such as metal foundries, constructions, and ceramic, quarry, and pottery industries. Since crystalline silica exposure has been linked with silicosis, lung cancer, and other pulmonary diseases, adverse effect attributed to this element has be a cause for concern worldwide. Silica dust exposure in workers is still considered to be important health problem especially in developing countries. The aim of the study was to investigate the effects of occupational silica exposure on oxidative stress parameters including the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and levels of total glutathione (GSH) and thiobarbituric acid reactive substance (TBARS) as well as immune system parameters such as interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, and IL-10 and tumor necrosis factor (TNF)-α in Turkish ceramic workers. In this study, nearly 50% of Turkish ceramic workers were diagnosed with silicosis. Eighty-four percent of these silicotic workers were found to present with profusion category 1 silicosis, whereas controls (n = 81) all displayed normal chest radiographs. Data demonstrated a significant decrease in levels of GSH and activities of CAT, SOD, and GPx, but a significant increase in MDA levels and activity of GR in all workers. Further, workers possessed significantly higher levels of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, and TNF-α. These observations suggest that ceramic workers may have impaired antioxidant/oxidant status and activated immune system indicative of inflammatory responses.

Assessment of Cytotoxicity Profiles of Different Phytochemicals: Comparison of Neutral Red and MTT Assays in Different Cells in Different Time Periods.

OBJECTIVES: Phenolic compounds exhibit several health protective properties. Galangin, curcumin, pycnogenol, puerarin and ursolic acid are commonly used plant phenolics in folk medicine. The aim of our study was to evaluate the difference between neutral red uptake (NRU) and MTT assays using different plant phenolics (galangin, curcumin, pycnogenol, puerarin and ursolic acid) in healthy and cancer cells in different time periods. MATERIALS AND METHODS: In this study, the cytotoxic effects of these phenolic compounds were investigated by NRU and MTT assays in healthy (V79, Chinese hamster fibroblast cell line) and cancer [human cervix epithelial adenocarcinoma cell line Henrietta Lacks (HeLa) and human mammary carcinoma cell line (BT-474)] in 18, 24 and 48 h incubation periods. RESULTS: Our results demonstrated that galangin, curcumin, pycnogenol, puerarin and ursolic acid decreased cell viability of V79, HeLa and BT-474 cells in a dose-dependent manner in 18, 24 and 48 h incubation periods. However, the cell survival rate was much lower in 48 h incubation period. There was no difference between the results from NRU and MTT assays. CONCLUSION: To decide which incubation period and which cytotoxicity study to be used, the cytotoxicity mechanism of the compound must be known.