by Lucia Luu, Nazanin Safavi and Antonio F. Machado Abstract Department of Environmental and Occupational Health, California State University, Northridge

BHA has been subjected to extensive toxicological study. Since there are emerging concerns about the possible If no antioxidant was used, the exposure of erythrocyte groups to BHA led to significant losses of influence of BHA on environmental and human health, this project focuses on the genotoxicity and phospholipids and cholesterol. So antioxidants have a potent inhibition effect on cholesterol carcinogenicity of BHA and potential risks from exposure. The genotoxicity of BHA has been studied in vitro and losses. Both antioxidants perform significant role in protecting phospholipids against losses. in vivo “which did not prove BHA to be mutagenic” (JECFA, 1987). BHA toxicity is low in rodents with an LD50 Cholesterol depletion in the erythrocyte membrane reduces the protective effects of antioxidants in mouse and rat > 2000 mg/kg bw/day. The International Agency for Research on Cancer (IARC) categorized against lipid peroxidation and ROS production. This will minimize the efficiency of antioxidants BHA as possibly carcinogenic to humans due to insufficient evidence in humans but sufficient evidence in against oxidative stress in cholesterol-depleted erythrocytes. (Sánchez-Gallego et al.).

animals. In the US National Toxicology Program Report on Carcinogens (NTP, 2005), BHA is predicted to be a carcinogen. However, based on the human population statistics, the usual low intake levels of BHA shows no significant association with an increased risk of cancer. Some experts recommend banning BHA, using instead BHA was also examined for its capacity to modulate the cytotoxic effects of ∆9 vitamin E or packing foods under nitrogen.

Background

tetrahydrocannabinol (THC). THC was not cytotoxic at low concentrations, but induced cell necrosis at higher levels. BHA, at concentrations of 10–200 _M, induced partial cell toxicity but considerably boosted the necrotic death resulting from concurrent exposure to THC. In the Butylated hydroxyanisole (BHA) consists of a mixture of two isomeric compounds, 2-

tert

-butyl-4 hydroxyanisole and 15% or less of 3-

tert

-butyl-4-hydroxyanisole. At room temperature, BHA is a white or presence of BHA at 200 _M, the LC50 for THC decreased. Neither of inhibitor compounds (diphenyleneiodonium or dicumarol ), protected cells from the effects of combined THC and slightly yellow, waxy solid with a faint characteristic odor (IARC 1986). BHA, but rather enhanced necrotic cell death. Both THC and BHA reduced ATP levels in A549 cells, and showed toxic effects on mitochondrial electron transport. The combination of BHA and

Property

Molecular weight 180.2a

Information

Emerging Water Contaminant: Butylated Hydroxyanisole

THC was synergistic in this respect, reducing ATP levels. Exposure to marijuana smoke in conjunction with BHA, may promote deleterious health effects in the lung. (Theodore A. Sarafian et al.) Melting point 48°C to 55°C

Effects of BHA in Aquatic Organisms

Boiling point 264°C to 270°C at 733 mm Hga http://ntp.niehs.nih.gov/go/roc13 Log Kow Water solubility Vapor pressure 3.5b

0.213 g/L at 25°Cb 0.00248 mm Hg at 25°Ca The use of BHA is very ubiquitous, so industrial wastewater and surface water containing BHA may effortlessly enter into the environment and disrupt the atmospheric, terrestrial, and aquatic ecosystems. BHA is known to have low soil mobility, volatilizes slowly from water, and has a high bioconcentration factor—these properties raise a concern that, over time, BHA may potentially bioaccumulate in the environment and in the tissues of organisms in the environment. Sources: aHSDB 2009, bChemIDplus 2009.

Commonly used in food, pharmaceuticals, cosmetics, and animal feed as an antioxidant and has also been found in water. Although deemed as a safe preservative, studies have found contradictory effects of BHA such as its role as an antioxidant and pro-oxidant, anti-carcinogen and carcinogen, and as an environmental endocrine disruptor (Nie et al.). Most studies have found various health risk outcomes from exposure to BHA. Health risks associated with oral ingestion of BHA include protection of animals against numerous xenobiotics by preventing carcinogenesis in numerous tissues such as the liver, lung, and breast (Vandghanooni et al.). In contrast, BHA has also been observed to act as a tumor initiator or tumor promoter and, at high doses, induces the formation of papillomas and carcinomas (Vandghanooni et al.).

Currently, very few literature has been published on the metabolic breakdown of xenobiotics in exposed algae—less than 1% of ecotoxicological data is available for pharmaceuticals (Sanderson et al.). The available studies, thus far, have assessed the behavioral response of phase I and phase II enzymes in algae as well as analyzing the potential ecotoxicological effects of BHA bioaccumulation (Nie et al.; Jos et al.) One of the few existing literature includes a study involving a battery with six different types of organisms and eighteen endpoints over a range of exposure times. Below is a table which summarizes the different organisms and respective endpoints of the study by Jos et al.

Mechanism of Action

BHA is oxidized to its metabolite,

tert

-butylhydroquinone (tBHQ), which is then further oxidized to 2-

tert

butyl(1,4)paraquinone (TBQ), a semiquinone anion radical and reactive oxygen species. Many studies have documented the cytotoxic effects of BHA exposure; however, the mechanism by which BHA induces cytotoxic effects is still relatively unclear. Some studies have suggested that the cytotoxic nature of BHA is dependent on the formation of its reactive intermediates while other studies have suggested that the cytotoxicity of BHA is mediated by the release of enzymatic constituents from mitochondria (Yu et al.). BHA-induced cytotoxicity seems to have occurred as a result of the metabolic activation of BHA to its metabolites, tBHQ and TBQ, by way of cytochrome P450 and peroxidases. The cytotoxicity of BHA metabolites on isolated rat hepatocytes may occur via a Cu(II)/Cu(I) redox cycle. The presence of Cu mediates the oxidation of tBHQ to TBQ, H 2 O 2 , and a hydroxyl radical, all of which induces DNA strand breaks (Y. Li et al.). Cu mediated oxidation of tBHQ to its reactive oxygen species and other constituents relies heavily upon the addition of Cu(II). Without Cu(II), the absence of a Cu(II)/Cu(I) redox cycle results in the absence of BHA-induced carcinogenesis. In contrast, studies conducted on rat hepatocytes have found that BHA may directly interact with mitochondria to induce apoptosis by initiating the release of caspase and cytochrome

c

(Yu et al.). The release of the enzymes trigger a series of events which induces cytotoxic effects in rat hepatocytes. A diagram of the proposed mechanism for BHA-induced cytotoxicity in rat hepatocytes is shown below.

Scavenging the reactive intermediates by antioxidants such as NAC or ascorbic acid did not affect BHA-induced apoptosis, suggesting that BHA alone may induce apoptosis. Also, the extent of the effects seem to be dose dependent. BHA exposure at the highest dose of 1 mM BHA seems to cause rapid damage to cell membrane as well as significant DNA fragmentation (Yu et al.). At doses of 250

u

M of BHA, rat hepatocytes were showing slight reductions in cell viability. In addition, enzymatic activities of caspase and cytochrome

c

significantly increased at doses of 500

u

M of BHA. The increase in the enzymatic activities seems to play a role in mediating the induction of apoptosis. In contrast, studies have shown the protective effects of BHA and another phenolic compound, Quercetin, against cellular injury induced by oxidative stress, when exposed to each antioxidant. There is evidence that indicates BHA is more protective than Quercetin against ROS generation, lipid peroxidation and lipid losses in oxidized erythrocytes. The protection against oxidative damage provided by both antioxidants decreased while membrane cholesterol depleted. The LC50 values were almost two-fold in the presence of quercetin as opposed to BHA in control. The study by Jos et al. found that exposure to BHA had various EC 50 1.2

u

M to more than 500

u

values ranging from m for 5, 15, and 60 minutes. The results of the study suggest that the most sensitive to the least sensitive organisms is as follows:

V. fischeri >D. magna > C. vulgaris >

Vero mammalian cell line >

A. cepa

(Jos et al.).

V. fischeri

seem to be the most sensitive to BHA and had immediately showed signs of bioluminescent decline after 15 minutes of BHA exposure. As for the rest of the organisms, most began showing apparent signs of BHA toxicity after 48 hours of BHA exposure.

The toxic effects of norfloxacin (NOR) and butylated hydroxyanisole (BHA) on the microalga Scene desmus obliquus were assessed in terms of growth rate. Scenedesmus obliquus is an ideal model aquatic organism. Growth of S. obliquus was decreased under high concentrations of BHA and NOR over the 96 h exposure period. Growth inhibition was 82.4% at 60.0 mg/l for NOR and 60.6% at 16.0 mg/l for BHA after 96 h. These types of assays, revealing toxic effects of NOR and BHA to phototrophs, could be employed to assess the potential risks of these xenobiotics to aquatic ecological systems. Increasing BHA concentration increased the inhibition of S. obliquus growth, displaying an obvious dose-response relationship (Nie et al.).

BHA was classified by the IARC as a possible carcinogen to humans according to the evidences in experimental animals. BHA and propylparaben (PPB) both are phenolic preservatives which have been reported to have possible impacts on environmental and human health. Though, the cytotoxicity of co-exposure to these compounds have not been discovered. This study, shows whether the BHA could stop the pro-oxidant effects of propylparaben in Vero cells. According to the results, concurrent exposure to both BHA and PPB induce greater cytotoxic effects and shows synergistic characteristics of their co-exposure. Binary mixture of these two chemicals caused G0/G1 cell cycle arrest as a result of oxidative stress and DNA double strand breaks. The evidences indicate that BHA stimulates the pro-oxidant effects of propylparaben (Lee et al.).

Animal Health Effects of BHA

Routes of human exposure to BHA are ingestion, inhalation, and dermal contact. The estimated average daily intake of BHA in the human diet was 4.3 mg (IARC, 1986). Workers are also potentially at risk of exposure to BHA in industries such as food production, and animal feed production, cosmetic manufacturers, and petroleum workers (Report on Carcinogens, 13 th edition). Currently, there has only been one epidemiological study which contained adequate data to evaluate the relationship between human cancer and BHA exposure. The population based nested case-control study of stomach cancer in men and woman within the Netherlands Cohort Study of dietary intake found no increase in risk at ordinary levels of dietary intake of BHA (Botterweck et al., 2000).

However, oral exposure to BHA have been observed to induce benign and malignant forestomach tumors in F334 rats, and the effects seem to be dose-related. There have also been a higher incidence of hyperplasia development in the forestomach in both male and female rats (Nobuyuki et al.). Male and female rats were orally fed a low or high dose of BHA, 98 and 414 mg/kg/day and 108 and 474 mg/kg/day, respectively, for a total of 112 weeks. Male and female rats treated with a higher dose of BHA had a significantly higher incident of developing papilloma and squamous cell carcinoma. BHA exposure has also been associated with acting as a weak endocrine disruptor and inducing cytotoxic effects on the reproductive system of rats such as reproductive function, maturation, and hormonal status before, during, and after fetus development. Exposure to BHA at 100 mg/kg or 500 mg/kg seems to decrease the weight of sex organs such as vagina, testes, and ventral prostate while increasing the weight of liver, adrenal and thyroid gland. Although the toxic effects seem to be weak, the decreased weight of sex organs, low steroid and thyroid hormone levels, and minor histopathological lesions of the thyroid gland give cause to suspect BHA as an endocrine disruptor (Jeong et al.).

A study which evaluated the estrogenic and androgenic activity of butylated hydroxyanisole (BHA), suggested the anti-estrogenic activity of BHA which decreases the uterine weight in immature female rat while it doesn’t have effect on the androgenic activity in castrated male rats. So many in vitro studies has also been performed about the endocrine disruptive trait of BHA. According to .Jeong et al. (2005), high dose BHA (500 mg/kg) can cause impaired reproductive functions such as delayed sexual maturation, and decreased vaginal weight. BHA 250 mg/kg also was shown that increased the TP-stimulated relative ventral prostate gland weight. Antioxidants were reported to have synergistic effects on testosterone in apoptosis of prostate cancer cells in castrated rats. High doses of BHA may induce forestomach carcinomas followed by cell injuries in rodents. Therefore BHA could be a rodent carcinogen. According to the evaluation of WHO (1989), BHA can cause hemorrhage, liver lesion, hyperplasia and hyperkeratosis in stomach mucosa in Rodents. (WHO, 1989; IARC, 1986; Botterweck et al., 2000). Body weight showed significant decrease while treated with BHA 250 and 500 mg/kg., but relative liver weight was increased at BHA 500 mg/kg.

Regulations

The 13 th Report on Carcinogens, published on October 2, 2014, listed current regulations for BHA. According to • the Food and Drug Administration (FDA), BHA is: Generally recognized as safe for use in food when the total of antioxidants is not greater than 0.2% of fat or • • • oil content. May be used as a food additive for direct addition to food intended for human consumption. May be used in manufacturing of food packaging materials as long as the amount does not exceed 0.005%. May be used as an antioxidant in defoaming agents for processed foods as long as the amount does not exceed 0.1% by weight of defoamer • • • In addition, the EPA has listed BHA as a contaminant on Candidate List 3. Contaminant candidates on this list: Currently not subjected to any proposed national primary drinking water regulation(s) Known or anticipated to occur in public water systems May require regulation under the Safe Drinking Water Act (SDWA )

Remediation

Currently, there is an absence of water treatment methods to remediate water sources in which BHA is found. As such, EPA has assigned BHA on the list of chemicals for Tier 1 Screening. BHA is identified under the Safe Drinking Water Act (SDWA) because the EPA believes this this chemical should be a candidate, at least for screening purposes, under EDSP testing because it may occur in sources of drinking water to which a substantial population may be exposed (EPA 2014).

Conclusions

Studies support the concept that BHA does not raise concern with respect to carcinogenicity and genotoxicity. BHA exposure, at high doses, has been shown to induce forestomach carcinomas in rodents. Since humans do not have forestomach, BHA does not pose as a hazard. BHA at lower concentrations such as 100 ppm, exert anticarcinogenic properties. Thus, an ADI of 0.5 mg/kg bw/day was established. Most of our studies concluded that BHA exposure had no association between rodent forestomach hyperplasia or human health effects. Therefore, authorities have recommended continued use of BHA (Iverson, 1995; JECFA, 1996). Based on these studies, BHA at levels found in consumer products is not significant enough to pose a risk to human health. 13.

14.

15.

16.

17.

18.

1.

2.

3.

4.

5.

6.

7.

References

8.

9.

10.

11.

12.

"EPA." Contaminant Candidate List 3. N.p., 8 Oct. 2009. Web. 15 Dec. 2014.

"National Toxicology Program." 13th Report on Carcinogens (RoC). N.p., 2 Oct. 2014. Web. 15 Dec. 2014.

A. Jos, G. Repetto, J.C. Rίos, A. del Peso, M. Salguero, M.J. Hazen, et al. Ecotoxicological evaluation of the additive butylated hydroxyanisole using a battery with six model systems and eighteen endpoints. Aquat Toxicol, 71 (2005), pp. 183–192 A.A.M, BOTTERWECK, Verhagen H, GOLDBOHM R. A, and KLEINJANS J. "Butylated Hydroxyanisole." Info. N.p., n.d. Web. 15 Dec. 2014.

EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), Scientific Opinion on the re-evaluation of butylated hydroxyanisole – BHA (E 320) as a food additive, EFSA Journal 2011;9(10):2392 Hwan Goo, Kanga,, Jeong Sang Hee, and Choa Joon Hyoung ,. "Download PDFs." Evaluation of Estrogenic and Androgenic Activity of Butylated Hydroxyanisole in Immature Female and Castrated Rats. N.p., n.d. Web. 15 Dec. 2014.

IARC. 1986. Butylated hydroxyanisole (BHA). In Some Naturally Occurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, vol. 40. Lyon, France: International Agency for Research on Cancer. pp. 123-159.

Ito, Nobuyuki, et al. "Carcinogenicity of butylated hydroxyanisole in F344 rats."Journal of the National Cancer Institute 70.2 (1983): 343-352.

Jeong, Sang-Hee, et al. "Effects of butylated hydroxyanisole on the development and functions of reproductive system in rats." Toxicology 208.1 (2005): 49-62 Li Y, Seacat A, Kuppusamy P, Zweier JL, Yager JD, Trush MA. Copper redox-dependent activation of 2-tert-butyl(1, 4)hydroquinone: formation of reactive oxygen species and induction of oxidative DNA damage in isolated DNA and cultured rat hepatocytes. Mutat Res. 2002b;518:123–33.

M, WILLIAMS*, G., McQUEEN C. A, and C. TONG. "Download PDFs." Toxicity Studies of Butylated Hydroxyanisole and Butylated Hydroxytoluene. II. Chronic Feeding Studies. American Health Foundation, n.d. Web. 15 Dec. 2014.

Sánchez-Gallego, Jose, Abel López-Revuelta, Jose L. Sardina,, and Guillermo López-Ruano. "Download PDFs." Comparative Antioxidant Capacities of Quercetin and Butylated Hydroxyanisole in Cholesterol-modified Erythrocytes Damaged by Tert-butylhydroperoxide. N.p., n.d. Web. 15 Dec. 2014 Sanderson H, Brain RA, Johnson DJ, Wilson CJ, Solomon KR (2004) Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular and sex hormones. Toxicology 203:27–40 Sarafian, Theodore A., Shaghig Kouyoumjian, Donald Tashkin, and Michael D. Roth. "Synergistic Cytotoxicity of 9-tetrahydrocannabinol and Butylated Hydroxyanisole." N.p., n.d. Web.

Somayeh Vandghanooni, Ali Forouharmehr, Morteza Eskandani, Abolfazl Barzegari, Vala Kafil, Soheila Kashanian, and Jafar Ezzati Nazhad Dolatabadi. DNA and Cell Biology. March 2013, 32(3): 98-103. doi:10.1089/dna.2012.1946.

WILLIAMS, G. M., M. J. IATROPOULOS1, and WHYSNER. "Download PDFs."

Safety Assessment of Butylated Hydroxyanisole and Butylated Hydroxytoluene as Antioxidant Food Additives

. N.p., n.d. Web. 15 Dec. 2014.

X. Nie, J. Gu, J. Lu, W. Pan, and Y. Yang, “Effects of norfloxacin and butylated hydroxyanisole on the freshwater microalga Scenedesmus obliquus,” Ecotoxicology, vol. 18, no. 6, pp. 677–684, 2009.

Yu R, Mandlekar S, Kong ANT (2000) Molecular mechanisms of Butylated hydroxyanisol- induced toxicity: induction of a poptosis through direct release of cytochrome c. Am Soci. Pharmacol Exp Ther 58: 431–437