Antioxidant; food additive; food preservative

By encouraging cytosolic calcium buildup and early endoplasmic reticulum in astrocytes, butylated hydroxyanisole causes neurotoxic effects [1]. Butylated hydroxyanisole (25-100 μM; 48 hours) causes mortality and inhibits the development of human astrocytes. Cell cycle-related protein expression is decreased and cell cycle inhibitory protein expression is increased when butylated hydroxyanisole (100 μM; 48 hours) is administered [1]. In NHA-SV40LT cells, butylated hydroxyanisole (100 μM; 48 hours) triggers signaling [1]. Additionally, pro-protein expression in the endoplasmic reticulum and cytosolic induction are enhanced by butylated hydroxyanisole [1]. Butylated hydroxyanisole stimulates the endoplasmic reticulum and modifies electrical currents to cause levels of tunneling cell malfunction [2]. Experiment on proliferation [1]

Butylated hydroxyanisole (200 mg/kg; ig; daily; for three consecutive days) generates distinct expression patterns of Nrf2 and detoxifying enzymes in the liver and small intestine of C57BL/6 [3].

---

Butylated hydroxyanisole (BHA) is widely used as an antioxidant and preservative in food, food packaging and medicines. Its chemopreventive properties are attributing to its ability to activate the transcription factor NF-E2 p45-related factor 2 (Nrf2), which directs central genetic programs of detoxification and protection against oxidative stress. This study was to investigate the histological changes of Nrf2 and its regulated phase II enzymes Nqo1, AKR1B8, and Ho-1 in wild-type (WT) and Nrf2(-/-) mice induced by BHA. The mice were given a 200mg/kg oral dose of BHA daily for three days. Immunohistochemistry revealed that, in the liver from WT mice, BHA increased Nqo1 staining in hepatocytes, predominately in the pericentral region. In contrast, the induction of AKR1B8 appeared mostly in hepatocytes in the periportal region. The basal and inducible Ho-1 was located almost exclusively in Kupffer cells. In the small intestine from WT mice, the inducible expression patterns of Nqo1 and AKR1B8 were nearly identical to that of Nrf2, with more intense staining in the villus than that the crypt. Conversely, Keap1 was more highly expressed in the crypt, where the proliferative cells reside. Our study demonstrates that BHA elicited differential expression patterns of phase II-detoxifying enzymes in the liver and small intestine from WT but not Nrf2(-/-) mice, demonstrating a cell type specific response to BHA in vivo.[3]

Astrocytes provide nutritional support, regulate inflammation, and perform synaptic functions in the human brain. Although butylated hydroxyanisole (BHA) is a well-known antioxidant, several studies in animals have indicated BHA-mediated liver toxicity, retardation in reproductive organ development and learning, and sleep deficit. However, the specific effects of BHA on human astrocytes and the underlying mechanisms are yet unclear. Here, we investigated the antigrowth effects of BHA through cell cycle arrest and downregulation of regulatory protein expression. The typical cell proliferative signaling pathways, phosphoinositide 3-kinase/protein kinase B and extracellular signal-regulated kinase 1/2, were downregulated in astrocytes after BHA treatment. BHA increased the levels of pro-apoptotic proteins, such as BAX, cytochrome c, cleaved caspase 3, and cleaved caspase 9, and decreased the level of anti-apoptotic protein BCL-XL. It also increased the cytosolic calcium level and the expression of endoplasmic reticulum stress proteins. Treatment with BAPTA-AM, a calcium chelator, attenuated the increased levels of ER stress proteins and cleaved members of the caspase family. We further performed an in vivo evaluation of the neurotoxic effect of BHA on zebrafish embryos and glial fibrillary acidic protein, a representative astrocyte biomarker, in a gfap:eGFP zebrafish transgenic model. Our results provide clear evidence of the potent cytotoxic effects of BHA on human astrocytes, which lead to disruption of the brain and nerve development.[1]

---

Butylated hydroxyanisole (BHA), a synthetic phenolic antioxidant (SPA), has been used as a food additive. However, BHA acts as an environmental hormone, i.e., endocrine disruptor. Here, we investigated BHA-induced male reproductive dysfunction in mouse Leydig and Sertoli cells. We found that BHA suppressed proliferation and induced cell cycle arrest in TM3 and TM4 cells. Furthermore, we investigated mitochondrial permeabilization, expression profiles of pro-apoptotic and anti-apoptotic proteins, calcium influx, and endoplasmic reticulum (ER) stress in testicular cells after BHA treatment. The results indicated that BHA-mediated calcium dysregulation and ER stress downregulated steroidogenesis- and spermatogenesis-related genes in mouse testis cell lines. Additionally, proliferation of both TM3 and TM4 cells in response to BHA treatment was regulated via the Mapk and Akt signaling pathways. Therefore, constant BHA exposure may lead to testicular toxicity via mitochondrial dysfunction, ER stress, and abnormal calcium levels in the testis[2].

Proliferation experiment [1]
Cell Types: NHA-SV40LT Cell
Tested Concentrations: 0 μM, 25 μM, 50 μM, 75 μM, 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Exhibits anti-proliferative effect.

---

Cell cycle analysis[1]
Cell Types: NHA-SV40LT Cell
Tested Concentrations: 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Downregulation of canonical cell proliferation signaling pathways, phosphoinositide 3-kinase/protein kinase B and extracellular signal-regulated kinase 1/2 .

---

Apoptosis analysis [1]
Cell Types: NHA-SV40LT Cell
Tested Concentrations: 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Increased levels of pro-apoptotic proteins (such as BAX, cytochrome c, cleaved caspase 3 and cleaved caspase 9), and Reduce levels of the anti-apoptotic protein BCL-XL.

---

Western Blot Analysis[1]
Cell Types: NHA-SV40LT Cell
Tested Concentrations: 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Increased expression of pro-apoptotic proteins and diminished levels of anti-apoptotic proteins. Asterisks indicate significant effects.

Animal/Disease Models: Fiveweeks old C57BL/6 mice (WT and Nrf2-/-) [3]
Doses: 200 mg/kg
Route of Administration: po (oral gavage), one time/day for three days
Experimental Results: Increased Nqo1 staining in hepatocytes , mainly in the area around the center.

Absorption, Distribution and Excretion
CONCENTRATIONS OF 2-TERT-BUTYL-4-METHOXYPHENOL & 2,2'-DIHYDROXY-3,3'-DI-TERT-BUTYL-5,5'DIMETHOXY-DIPHENYL (DI-BHA) APPEARED AT DIFFERENT TIMES (0.15-24 HR) IN RAT PLASMA & INTESTINE FOLLOWING 2 G/KG SINGLE ORAL ADMIN OF 2-TERT-BUTYL-4-METHOXYPHENOL. PEAK CONCN IN ALL TISSUES ANALYZED WERE OBSERVED WITHIN 1 HR OF ADMIN. IN INTESTINE 2-TERT-BUTYL-4-METHOXYPHENOL LEVELS WERE APPROX 10 TIMES HIGHER THAN DI-BHA; IN PLASMA THEY WERE BETWEEN 100 & 15 TIMES HIGHER. THE RAT INTESTINE IS CAPABLE OF TRANSFORMING IN VIVO 2-TERT-BUTYL-4-METHOXYPHENOL INTO DI-BHA & MAY BE MAJOR SITE WHERE THIS TRANSFORMATION OCCURS.
Absorption of BHA from the digestive tract is by passive diffusion.
BHA was fed to groups of three beagle dogs at dose levels of 0, 0.3, 3, 30 and 100 mg/kg bw for 1 yr. All animals survived; no pathological lesion was seen, and there was not demonstrable storage of BHA ... .
In male and female Sprague-Dawley rats, BHA was absorbed rapidly after oral administration and metabolized.
When male volunteers were given an oral dose of 50 mg BHA, 27-77% was excreted in the urine as the glucuronide and urinary metabolites. ... Urinary excretion of BHA was maximal within 17 hr and complete by 48 hr. ... When human volunteers were given a single oral dose of 14(C)-labelled BHA (approx 0.5 mg/kg bw), 60-70% of the radioactivity was excreted in the urine within 2 days and 80-86.5% by day 11. ... Four male volunteers were given 30 mg BHA orally and 10 days later

---

Metabolism / Metabolites
FOLLOWING ORAL ADMIN OF 2 G/KG OF 2-TERT-BUTYL-4-METHOXYPHENOL TO RATS, THE METABOLITE 2,2'-DIHYDROXY-3,3'-DI-TERT-BUTYL-5,5'-DIMETHOXYDIPHENYL WAS DETECTED IN PLASMA & TISSUE 0.15-24 HR AFTER ADMIN.
DI-BHA (2,2'-DIHYDROXY-3,3'-DI-TERT-BUTYL-5,5'-DIMETHOXYDIPHENYL) WAS ISOLATED AS REACTION PRODUCT OF EITHER COMMERCIAL HORSERADISH PEROXIDASE OR PARTIALLY PURIFIED RAT INTESTINE PEROXIDASE & HYDROGEN PEROXIDE WITH 2-TERT-BUTYL-4-METHOXYPHENOL. CYCLIC COMPOUNDS (SUCH AS DI-BHA) POSSESSING A HYDROXY GROUP IN THE RING ARE COMPETITIVE INHIBITORS WITH RESPECT TO GUAIACOL & NON-COMPETITIVE INHIBITORS WITH RESPECT TO HYDROGEN PEROXIDASE IN A SYSTEM CONTAINING GUAIACOL, HYDROGEN PEROXIDASE & PEROXIDASE.
In rat, oral doses (0.4 g/kg) are excreted largely in urine, as glucuronide conjugate (72% of dose) with smaller ammounts of ethereal sulfate (14%) and of unchanged BHA (5%). Similar pattern of metabolism is ... seen in rabbit and human ...
... Dogs excrete only small amounts of BHA glucuronide in urine (5.5%) and most of dose is excreted as unchanged BHA in feces. Dogs also excrete greater proportion as ethereal sulfate (23% of dose), and ... form hydroxylated and demethylated metabolites ... not detected in human urine.
In male and female Sprague-Dawley rats, BHA was absorbed rapidly after oral administration and metabolized. The main metabolites were 4-O-conjugates: the O-sulfate and the O-glucuronide ... (No data on absorption via the skin were available to the Working Group.)
After administration of a single oral dose of 1000 mg BHA to New Zealand white rabbits, 46% of the dose was excreted in the urine as glucuronides, 9% as ethereal sulfates and 6% as free phenols. Excretion of glucuronides was inversely dose dependent: 60% was recovered as glucuronides after a dose of 500 mg and 84% after 250 mg. Recovery of BHA as glucuronide after repeated dosing (three or four doses) was lower than that after a single dose ... .

Toxicity Summary
IDENTIFICATION AND USE: 3-tert-Butyl-4-hydroxyanisole is a component of Butylated Hydroxyanisole which is a commonly used food preservative. This material is an oxidation inhibitor and has been accepted for the use in foods where Butylated hydroxytoluene is restricted. This chemical is used as a food preservative for human consumption. HUMAN EXPOSURE AND TOXICITY: There is no human studies concerning.3-tert-Butyl-4-hydroxyanisole ANIMAL STUDIES: The mutagenicity of 3-tert-butyl-4-hydroxyanisole and its metabolites was determined in the reverse mutation assay using Salmonella typhimurium, strains and in the chromosomal aberration test in vitro using the Chinese hamster fibroblast cell line, the chemical did not show any mutagenic activity. 3-tert-Butyl-4-hydroxyanisole induced chromosomal aberrations only in the presence of S9 mix.
IDENTIFICATION AND USE: Butylated hydroxyanisole (BHA) is white or slightly yellow, waxy solid. The primary use for BHA is as an antioxidant and preservative in food, food packaging, animal feed, and cosmetics, and rubber and petroleum products as well. BHA is particularly useful in protecting the flavors and color of essential oils and is considered the most effective of all food-approved antioxidants for this application. BHA is effective in controlling the oxidation of short-chain fatty acids, such as those found in coconut and palm kernel oils that are used typically in cereal and confectionary products. HUMAN EXPOSURE AND TOXICITY: BHA produced allergic contact dermatitis. Although BHA is not an irritant, it can cause skin reactions owing to its weak allergic sensitization potential. BHA was able to reduce DNA damage and micronucleus formed in peripheral lymphocytes exposed to other genotoxic agents. ANIMAL STUDIES: Dietary exposure to BHA caused benign and malignant tumors of the forestomach (papilloma and squamous-cell carcinoma) in rats of both sexes and in male mice and hamsters due to increased lifespan of the animals compared to control. Rats could tolerate BHA up to 1200 ppm in their diet for a period of 21 months. No histopathological or carcinogenic effects were found to be associated with the ingestion, nor were there any affects on reproduction. BHA was evaluated in rats and mice in dose levels up to 1000 and 500 mg/kg, respectively, for teratological effects. No abnormal findings attributable to the exposure were noted. In another study the teratological effect of BHA were evaluated in rabbits at dose levels ranging from 50 to 400 mg/kg. No abnormal findings were reported. Dietary administration of BHA to fish (hermaphroditic Rivulus marmoratus) as larvae caused liver cancer (hepatocellular carcinoma) in the adult fish. BHA at levels ranging from 5 to 500 mg/kg produced neurological changes in mice by increasing serotonin utilization in the central nervous system. ECOTOXICITY STUDIES: Dietary BHA produced alterations in the enzyme activities of the mixed-function oxidase system, changes in the ethyl isocyanide binding ratio and decreases in cytochrome P-450 content in the hepatic microsomes of rainbow trout.

---

Toxicity Summary
IDENTIFICATION AND USE: 3-tert-Butyl-4-hydroxyanisole is a component of Butylated Hydroxyanisole which is a commonly used food preservative. This material is an oxidation inhibitor and has been accepted for the use in foods where Butylated hydroxytoluene is restricted. This chemical is used as a food preservative for human consumption. HUMAN EXPOSURE AND TOXICITY: There is no human studies concerning.3-tert-Butyl-4-hydroxyanisole ANIMAL STUDIES: The mutagenicity of 3-tert-butyl-4-hydroxyanisole and its metabolites was determined in the reverse mutation assay using Salmonella typhimurium, strains and in the chromosomal aberration test in vitro using the Chinese hamster fibroblast cell line, the chemical did not show any mutagenic activity. 3-tert-Butyl-4-hydroxyanisole induced chromosomal aberrations only in the presence of S9 mix.

IDENTIFICATION AND USE: Butylated hydroxyanisole (BHA) is white or slightly yellow, waxy solid. The primary use for BHA is as an antioxidant and preservative in food, food packaging, animal feed, and cosmetics, and rubber and petroleum products as well. BHA is particularly useful in protecting the flavors and color of essential oils and is considered the most effective of all food-approved antioxidants for this application. BHA is effective in controlling the oxidation of short-chain fatty acids, such as those found in coconut and palm kernel oils that are used typically in cereal and confectionary products. HUMAN EXPOSURE AND TOXICITY: BHA produced allergic contact dermatitis. Although BHA is not an irritant, it can cause skin reactions owing to its weak allergic sensitization potential. BHA was able to reduce DNA damage and micronucleus formed in peripheral lymphocytes exposed to other genotoxic agents. ANIMAL STUDIES: Dietary exposure to BHA caused benign and malignant tumors of the forestomach (papilloma and squamous-cell carcinoma) in rats of both sexes and in male mice and hamsters due to increased lifespan of the animals compared to control. Rats could tolerate BHA up to 1200 ppm in their diet for a period of 21 months. No histopathological or carcinogenic effects were found to be associated with the ingestion, nor were there any affects on reproduction. BHA was evaluated in rats and mice in dose levels up to 1000 and 500 mg/kg, respectively, for teratological effects. No abnormal findings attributable to the exposure were noted. In another study the teratological effect of BHA were evaluated in rabbits at dose levels ranging from 50 to 400 mg/kg. No abnormal findings were reported. Dietary administration of BHA to fish (hermaphroditic Rivulus marmoratus) as larvae caused liver cancer (hepatocellular carcinoma) in the adult fish. BHA at levels ranging from 5 to 500 mg/kg produced neurological changes in mice by increasing serotonin utilization in the central nervous system. ECOTOXICITY STUDIES: Dietary BHA produced alterations in the enzyme activities of the mixed-function oxidase system, changes in the ethyl isocyanide binding ratio and decreases in cytochrome P-450 content in the hepatic microsomes of rainbow trout.

---

Non-Human Toxicity Values
LD50 Rat oral 4000 mg/kg

LD50 Rabbit oral 2100 mg/kg

LD50 Mouse oral 2000 mg/kg bw

---

Interactions
IN TESTS ON BENZO(A)PYRENE-INDUCED NEOPLASIA OF FORESTOMACH OF ICR/HA MOUSE, ADDN OF 3-TERT-BUTYL-4-HYDROXYANISOLE DECR NUMBER & INCIDENCE OF TUMOR FORMATION.
MICE MAINTAINED ON DIET CONTAINING 0.75% 2(3)-TERT-BUTYL-4-HYDROXYANISOLE (BHA) FOR 8 DAYS SHOWED 50% REDUCTION IN MAXIMAL ORNITHINE DECARBOXYLASE (ODC) INDUCTION FOLLOWING TREATMENT WITH TPA (12-O-TETRADECANOYLPHORBOL-13-ACETATE) WHEN COMPARED TO MICE FED CONTROL DIET. TOPICAL APPLICATION OF BHA (55 UMOLE) 30 MIN PRIOR TO TPA TREATMENT (17 NMOL) ELICITED 80% INHIBITION OF PROMOTER-INDUCED ODC ACTIVITY. BHA WAS INEFFECTIVE AS INHIBITOR WHEN ADMIN 16 HR BEFORE OR 2 HR AFTER THE PROMOTER. INHIBITION WAS DOSE DEPENDENT WITH A DOSE PRODUCING 50% INHIBITION OF ODC INDUCTION OF 6 UMOLE. A STRUCTURE-ACTIVITY STUDY SHOWED THAT HYDROXYL & TERT-BUTYL SUBSTITUENTS WERE IMPORTANT DETERMINANTS OF INHIBITORY ACTIVITY.
RATS WERE FED 2(3)-TERT-BUTYL-4-HYDROXYANISOLE (0.5% WT/WT) IN DIET WITH OR WITHOUT CIPROFIBRATE (10 MG/KG BODY WT) FOR 60 WK. 2(3)-TERT-BUTYL-4-HYDROXYANISOLE CAUSED SIGNIFICANT DECR IN INCIDENCE & NUMBER OF HEPATOCELLULAR CARCINOMAS THAT WERE LARGER THAN 5 MM. DATA SUGGEST THAT INHIBITORY EFFECT ON CIPROFIBRATE-INDUCED HEPATIC TUMORIGENESIS MAY BE DUE TO H2O2 & FREE RADICAL-SCAVENGING PROPERTY, SINCE IT DOES NOT PREVENT PEROXISOMES PROLIFERATION & INDUCTION OF H2O2-GENERATING PEROXISOMAL ENZYMES IN LIVERS OF RATS FED CIPROFIBRATE.
BHA LD50 was determined in mice and rats following ip or oral admin, using dimethylsulfoxide or olive oil as vehicle. When dimethylsulfoxide was used, BHA ip LD50 was about two orders of magnitude lower as compared to oral LD50. This difference was not so marked when BHA was administered in olive oil. /BHA/
/Both BHA and BHT have chemopreventive action when/ administered together with several carcinogens affecting diverse target organs. The antioxidants increase detoxifying enzymes for carcinogens, and many act as free radical trapping agents. The structure of these agents does not suggest a likely electrophile and tests for genotoxicity have been uniformly negative. Both BHA and BHT have been shown to inhibit cell-to-cell communication in culture. /BHA/
Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. /The objective of the study was/ to determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.
Butylated hydroxyanisole and propylparaben are phenolic preservatives commonly used in food, pharmaceutical and personal care products. Both chemicals have been subjected to extensive toxicological studies, due to the growing concern regarding their possible impacts on environmental and human health. However, the cytotoxicity and underlying mechanisms of co-exposure to these compounds have not been explored. In this study, a set of relevant cytotoxicity endpoints including cell viability and proliferation, oxidative stress, DNA damage and gene expression changes were analyzed to assess whether the antioxidant butylated hydroxyanisole could prevent the pro-oxidant effects caused by propylparaben in Vero cells. We demonstrated that binary mixtures of both chemicals induce greater cytotoxic effects than those reported after single exposure to each compound. Simultaneous treatment with butylated hydroxyanisole and propylparaben caused G0/G1 cell cycle arrest as a result of enhanced generation of oxidative stress and DNA double strand breaks. DNA microarray analysis revealed that a cross-talk between transforming growth factor beta (TGFbeta) and ataxia-telangiectasia mutated kinase (ATM) pathways regulates the response of Vero cells to the tested compounds in binary mixture. Our findings indicate that butylated hydroxyanisole potentiates the pro-oxidant effects of propylparaben in cultured mammalian cells and provide useful information for their safety assessment.
The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT and BHA+BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing.
The present study was aimed to study protective effect of butylated hydroxyanisole (BHA), a phenolic antioxidant used in foods on ferric nitrilotriacetate (Fe-NTA)-induced nephrotoxicity. Male albino rats of Wistar strain (4-6 weeks old) weighing 125-150 g were used in this study. Animals were given a single dose of Fe-NTA (9 mg/kg body weight) after treatment with BHA (1 and 2 mg/animal/day). Fe-NTA treatment enhanced ornithine decarboxylase (ODC) activity to 5.3-fold, and [(3)H]-thymidine incorporation in DNA to 2.5-fold in kidney compared with the corresponding saline-treated control, whereas glutathione (GSH) levels and the activities of antioxidant enzymes decreased to a range of 2- to 2.5-fold in kidney. These changes were reversed significantly in animals receiving a pretreatment of BHA. The enhanced ODC activity and DNA synthesis showed a reduction to 2.12-fold and 1.15-fold, respectively, at a higher dose of 2 mg BHA/day/animal, compared with the Fe-NTA-treated groups. Pretreatment with BHA prior to Fe-NTA treatment increased GSH and the activities of antioxidant enzymes to a range of 1.5- to 2-fold in kidney. The results indicate that BHA suppresses Fe-NTA-induced nephrotoxicity in male Wistar rats.
For more Interactions (Complete) data for Butylated hydroxyanisole (32 total), please visit the HSDB record page.

---

Non-Human Toxicity Values
LD50 Rat oral 4000 mg/kg
LD50 Rabbit oral 2100 mg/kg
LD50 Mouse oral 2000 mg/kg bw
LD50 Rat oral 2200 mg/kg bw
LD50 Rat (male) ip 881 mg/kg bw

[1]. Sunwoo Park, et al. Butylated Hydroxyanisole Exerts Neurotoxic Effects by Promoting Cytosolic Calcium Accumulation and Endoplasmic Reticulum Stress in Astrocytes. J Agric Food Chem. 2019 Aug 28;67(34):9618-9629.
[2]. Jiyeon Ham, et al. Butylated Hydroxyanisole Exerts Neurotoxic Effects by Promoting Cytosolic Calcium Accumulation and Endoplasmic Reticulum Stress in Astrocytes. Sci Total Environ. 2020 Feb 1;702:134775.
[3]. Lin Luo, et al. Butylated hydroxyanisole induces distinct expression patterns of Nrf2 and detoxification enzymes in the liver and small intestine of C57BL/6 mice. Toxicol Appl Pharmacol. 2015 Nov 1;288(3):339-48.
[4]. Jennifer Yinuo Cao, et al. Mechanisms of ferroptosis. Cell Mol Life Sci. 2016 Jun;73(11-12):2195-209.

Butylated hydroxyanisole appears as white, beige or slightly yellow waxy solid with an aromatic odor and a slightly bitter burning taste. (NTP, 1992)
3-tert-butyl-4-hydroxyanisole is an aromatic ether that is 4-methoxyphenol in which one of the hydrogens ortho- to the phenolic hydroxy group is replaced by a tert-butyl group. It has a role as an antioxidant and a human xenobiotic metabolite. It is a member of phenols and an aromatic ether.
2-tert-Butyl-4-methoxyphenol has been reported in Salvia officinalis, Murraya paniculata, and Dillenia indica with data available.

---

Mechanism of Action
ADMIN OF 2(3)-TERT-BUTYL-4-HYDROXYANISOLE (BHA) TO RODENTS PROTECTS A VARIETY OF TARGET TISSUES AGAINST PRODUCTION OF TUMORS BY WIDE RANGE OF CHEMICAL CARCINOGENS. BHA REDUCES LEVELS OF MUTAGENIC METABOLITES PRODUCED FROM BENZO(A)PYRENE & NUMEROUS THERAPEUTIC AGENTS IN VIVO; IT ELEVATES HEPATIC ACTIVITIES OF MICROSOMAL EPOXIDE HYDRATASE & CYTOSOL GLUTATHIONE S-TRANSFERASE; IT ALTERS ACTIVITIES OF OTHER HEPATIC ENZYMES & AFFECTS LEVELS OF SOME HEPATIC CATALYTIC CONSTITUENTS; & IT INCREASES CONCN OF NONPROTEIN THIOL COMPOUNDS IN LIVER & SEVERAL OTHER TISSUES. /BHA/
EFFECTS OF BUTYLATED HYDROXYANISOLE (BHA) ON ARYL HYDROCARBON HYDROXYLASE (AHH) ACTIVITES IN LIVER, LUNG & SKIN OF RATS & MICE WERE STUDIED TO EXAMINE POSSIBLE MECHANISMS OF ANTICARCINOGENIC ACTIONS. AHH INDUCERS, 3-METHYLCHOLANTHRENE, PHENOBARBITAL &2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN WERE USED. IT WAS OBSERVED THAT 2-TERT-BUTYL-4-HYDROXYANISOLE, 3-TERT-BUTYL-4-HYDROXYANISOLE & BHA COMMERCIAL MIXTURE (85% 3-BHA & 15% 2-BHA) HAD ABOUT THE SAME POTENCY IN INHIBITING MICROSOMAL AHH, ALTHOUGH THE 2-ISOMER APPEARED TO BE SLIGHTLY MORE INHIBITORY. DATA SUGGEST THAT INHIBITORY ACTION OBSERVED IN COMMERCIAL BHA IS DUE TO THE COMBINED ACTION OF BOTH ISOMERS & IS DEPENDENT ON SPECIES OF ANIMAL, TISSUE TYPES & TREATMENT WITH INDUCERS.
/Both BHA and BHT have chemopreventive action when/ administered together with several carcinogens affecting diverse target organs. The antioxidants increase detoxifying enzymes for carcinogens, and many act as free radical trapping agents. The structure of these agents does not suggest a likely electrophile and tests for genotoxicity have been uniformly negative. Both BHA and BHT have been shown to inhibit cell-to-cell communication in culture. /BHA/
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17beta-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis. Female ACI rats were treated with E2; Vit C; Vit C+E2; BHA; and BHA+E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2. The expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage. Our studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.

C11H16O2

180.2435

360.23

25013-16-5

8456

White or slightly yellow waxy solid

264-270 ºC

48-63 ºC

130 ºC

3.2

1

2

2

13

160

0

O([H])C1C([H])=C([H])C(=C([H])C=1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H]

MRBKEAMVRSLQPH-UHFFFAOYSA-N

InChI=1S/C11H16O2/c1-11(2,3)9-7-8(13-4)5-6-10(9)12/h5-7,12H,1-4H3

2-tert-butyl-4-methoxyphenol

2-tert-Butyl-4-methoxyphenol; 3-tert-Butyl-4-hydroxyanisole; 121-00-6; 25013-16-5; 4-Hydroxy-3-tert-butylanisole; 2-(tert-butyl)-4-methoxyphenol; 3-BHA; 3-T-BUTYL-4-HYDROXYANISOLE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~554.82 mM)
H2O : ~1 mg/mL (~5.55 mM)

Solubility in Formulation 1: 100 mg/mL (554.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)