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]

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In primary rat astrocytes, Butylhydroxyanisole (BHA) (10-100 μM) induced concentration-dependent cytotoxicity. After 24-hour treatment, cell viability (MTT assay) was reduced by 23% (10 μM), 47% (50 μM), and 78% (100 μM), with an IC50 of 42.3 μM. It promoted cytosolic calcium ([Ca²⁺]c) accumulation (2.1-fold increase at 50 μM, detected by Fura-2 AM staining) and endoplasmic reticulum (ER) stress, as evidenced by upregulated GRP78 (2.8-fold), CHOP (3.5-fold), and phosphorylated eIF2α (2.3-fold) via western blot [1]
In primary mouse astrocytes, Butylhydroxyanisole (BHA) (25-100 μM, 24 hours) caused similar [Ca²⁺]c elevation (maximal 2.4-fold at 100 μM) and ER stress. It activated caspase-3 (cleaved form upregulated 3.1-fold) and induced apoptotic cell death (Annexin V-FITC/PI staining: 28% apoptotic rate at 100 μM). Pretreatment with an ER stress inhibitor (4-PBA, 10 μM) or calcium chelator (BAPTA-AM, 5 μM) reversed cytotoxicity by 45% and 52% respectively [2]

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].

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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]

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In male C57BL/6 mice (6-8 weeks old) fed diets containing 0.1% or 0.4% (w/w) Butylhydroxyanisole (BHA) for 14 days, hepatic Nrf2 protein expression was upregulated by 1.8-fold (0.1% dose) and 2.5-fold (0.4% dose) compared to the control. Hepatic detoxification enzymes CYP1A1 (1.7-fold), CYP2E1 (1.5-fold), and GST (2.2-fold) were significantly induced at the 0.4% dose [3]
In the small intestine of the same mice, Butylhydroxyanisole (BHA) did not alter Nrf2 expression but increased GST activity by 1.4-fold (0.4% dose) and UDP-glucuronosyltransferase (UGT) by 1.3-fold (0.4% dose). No significant changes in liver/intestinal histology or serum ALT/AST levels were observed [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]

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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.

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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 .

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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.

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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.

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Primary rat astrocytes were isolated from neonatal rat brains and cultured in DMEM supplemented with fetal bovine serum. Cells were seeded in 96-well plates (5×10³ cells/well) or 6-well plates (2×10⁵ cells/well) and incubated for 72 hours to adhere. Butylhydroxyanisole (BHA) was dissolved in DMSO and added at concentrations of 10-100 μM. After 24-hour treatment, cell viability was measured by MTT assay; [Ca²⁺]c was detected via Fura-2 AM fluorescence imaging; ER stress markers (GRP78, CHOP, p-eIF2α) were quantified by western blot [1]
Primary mouse astrocytes were cultured in neurobasal medium. Cells were treated with Butylhydroxyanisole (BHA) (25-100 μM) for 24 hours. Apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry; caspase-3 activation was detected by western blot. For rescue experiments, cells were pretreated with 4-PBA (10 μM) or BAPTA-AM (5 μM) for 1 hour before BHA administration [2]

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.

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Male C57BL/6 mice (6-8 weeks old, 20-25 g) were randomly divided into control, 0.1% BHA, and 0.4% BHA groups (n=6 per group). Butylhydroxyanisole (BHA) was mixed into standard rodent chow at concentrations of 0.1% and 0.4% (w/w). Mice were fed the respective diets ad libitum for 14 days; control mice received standard chow without BHA. At the end of the experiment, mice were euthanized, and liver and small intestine tissues were collected. Tissues were homogenized for Nrf2 protein detection (western blot) and detoxification enzyme activity/expression analysis (qPCR and enzymatic assays) [3]

Absorption, Distribution and Excretion
Following a single oral administration of 2 g/kg 2-tert-Butylated-4-methoxyphenol to rats, concentrations of 2-tert-Butylated-4-methoxyphenol and 2,2'-dihydroxy-3,3'-di-tert-Butylated-5,5'-dimethoxydiphenyl (DI-BHA) appeared in rat plasma and intestine at different time points (0.15–24 hours). Peak concentrations in all analyzed tissues were observed within 1 hour of administration. In the intestine, the concentration of 2-tert-Butylated-4-methoxyphenol was approximately 10 times that of diButylatedhydroxyanisole (DI-BHA); in plasma, the concentration was 100 to 15 times higher. The rat intestine is capable of converting 2-tert-Butylated-4-methoxyphenol to DI-BHA in vivo and is likely the primary site of this conversion. BHA absorption from the digestive tract occurs via passive diffusion.
BHA was administered to three groups of beagle dogs at dose levels of 0, 0.3, 3, 30, and 100 mg/kg body weight for one year. All animals survived; no pathological damage or significant accumulation of BHA was observed…
In male and female Sprague-Dawley rats, BHA was rapidly absorbed and metabolized after oral administration.
In male volunteers, after oral administration of 50 mg BHA, 27-77% was excreted in the urine as glucuronide and urinary metabolites. …Urinary excretion of BHA peaked within 17 hours and was completely eliminated within 48 hours. …When human volunteers received a single oral dose of 14C-labeled BHA (approximately 0.5 mg/kg body weight), 60-70% of the radioactive material was excreted in the urine within 2 days, and 80-86.5% was eliminated by day 11. …Four male volunteers were orally administered 30 mg of BHA. Ten days later, the metabolites were detected in plasma and tissues in rats after oral administration of 2 g/kg 2-tert-Butylated-4-methoxyphenol, within 0.15–24 hours post-administration. DI-BHA (2,2'-dihydroxy-3,3'-di-tert-Butylated-5,5'-dimethoxydiphenyl) is a product of the reaction of commercial horseradish peroxidase or partially purified rat intestinal peroxidase and hydrogen peroxide with 2-tert-Butylated-4-methoxyphenol. Cyclic compounds (e.g., Butylatedated hydroxyanisole, DI-BHA) contain a hydroxyl group on their ring and are competitive inhibitors of guaiacol and non-competitive inhibitors of catalase in systems containing guaiacol, hydrogen peroxide, and peroxidase. In rats, the oral dose (0.4 g/kg) was primarily excreted in the urine as glucuronide conjugates (72% of the dose), with smaller amounts excreted as ethyl sulfate (14%) and unmetabolized BHA (5%). Similar metabolic patterns were observed in rabbits and humans… Dogs excrete only a small amount of BHA glucuronide (5.5%) in the urine, with the majority of the dose excreted in the feces as unmetabolized BHA. Canine excretion of BHA is primarily in the form of ether sulfate (23% of the dose) and forms hydroxylated and demethylated metabolites, which are not detected in human urine. In male and female Sprague-Dawley rats, oral BHA is rapidly absorbed and metabolized. The main metabolites are 4-O-conjugates: O-sulfate and O-glucuronide… (The working group did not obtain data on transdermal absorption.)
After a single oral dose of 1000 mg BHA in New Zealand white rabbits, 46% of the dose was excreted in the urine as glucuronide, 9% as ether sulfate, and 6% as free phenol. The excretion of glucuronide was inversely proportional to the dose: after a 500 mg dose, 60% was excreted as glucuronide; after a 250 mg dose, 84% was excreted as glucuronide. After repeated administration (three or four times), the amount of BHA recovered as glucuronide was lower than that recovered after a single dose…

Toxicity Summary
Identification and Uses: 3-tert-Butylated-4-hydroxyanisole is a component of Butylatedated hydroxyanisole, a commonly used food preservative. This substance is an oxidation inhibitor and has been approved for use in foods where Butylatedated hydroxytoluene is restricted. This chemical is used as a food preservative for human consumption. Human Exposure and Toxicity: There are currently no human studies on 3-tert-Butylated-4-hydroxyanisole. Animal Studies: The mutagenicity of 3-tert-Butylated-4-hydroxyanisole and its metabolites was determined using reverse mutation assays with Salmonella typhimurium strains and in vitro chromosomal aberration assays with Chinese hamster fibroblast cell lines. The results showed that this chemical did not exhibit any mutagenic activity. 3-tert-Butylated-4-hydroxyanisole induces chromosomal aberrations only in the presence of S9 mixtures. Identification and Uses: Butylatedated hydroxyanisole (BHA) is a white or slightly yellow waxy solid. The primary uses of BHA are as an antioxidant and preservative in food, food packaging, animal feed, cosmetics, and rubber and petroleum products. BHA is particularly effective at protecting the flavor and color of essential oils and is considered the most potent of all food-grade antioxidants. BHA effectively controls the oxidation of short-chain fatty acids, such as those found in coconut oil and palm kernel oil, which are commonly used in cereal and confectionery products. Human Exposure and Toxicity: BHA can cause allergic contact dermatitis. Although BHA itself is not irritating, it may still cause skin reactions due to its weak sensitizing properties. BHA can reduce DNA damage and micronucleus formation in peripheral lymphocytes exposed to other genotoxic substances. Animal Studies: Compared to control groups, male and female rats, as well as male mice and hamsters, experienced prolonged lifespans and developed benign and malignant tumors (papilloma and squamous cell carcinoma) in their forestomach. Rats tolerated dietary BHA up to 1200 ppm for up to 21 months. No histopathological or carcinogenic effects related to intake were observed, nor were any effects on reproduction found. The teratogenic effects of BHA were assessed in rats and mice at dose levels up to 1000 and 500 mg/kg, respectively. No abnormal results were observed related to exposure. In another study, the teratogenic effects of BHA in rabbits were assessed at dose levels ranging from 50 to 400 mg/kg. No abnormal results were reported. Feeding BHA to juvenile fish (hermaphroditic killifish) led to hepatocellular carcinoma in adult fish. BHA doses in the range of 5 to 500 mg/kg increased serotonin utilization in the central nervous system of mice, resulting in neurological changes. Ecotoxicity studies: Feeding BHA resulted in altered enzyme activity of the mixed-function oxidase system in rainbow trout liver microsomes, altered ethyl isocyanate binding, and decreased cytochrome P-450 content.

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Toxicity Overview
Identification and Uses: 3-tert-Butylated-4-hydroxyanisole is a component of Butylatedated hydroxyanisole, a commonly used food preservative. This substance is an oxidation inhibitor and has been approved for use in foods where the use of Butylatedated hydroxytoluene (BHA) is restricted. This chemical is used as a food preservative for human consumption. Human exposure and toxicity: There are currently no human studies on 3-tert-Butylated-4-hydroxyanisole. Animal studies: The mutagenicity of 3-tert-Butylated-4-hydroxyanisole and its metabolites was determined using reverse mutagenesis assays with Salmonella Typhimurium strains and in vitro chromosomal aberration assays with Chinese hamster fibroblast cell lines. The results showed that this chemical did not exhibit any mutagenic activity. 3-tert-Butylated-4-hydroxyanisole induces chromosomal aberrations only in the presence of S9 mixtures. Identification and uses: Butylatedhydroxyanisole (BHA) is a white or slightly yellow waxy solid. The main uses of BHA are as an antioxidant and preservative in food, food packaging, animal feed, cosmetics, and rubber and petroleum products. BHA is particularly effective in protecting the flavor and color of essential oils and is considered the most potent of all food-grade antioxidants. BHA effectively controls the oxidation of short-chain fatty acids, such as those found in coconut oil and palm kernel oil, which are commonly used in cereals and confectionery products. Human exposure and toxicity: BHA can cause allergic contact dermatitis. Although BHA itself is not irritating, it may still cause skin reactions due to its weak sensitizing properties. BHA can reduce DNA damage and micronucleus formation in peripheral lymphocytes exposed to other genotoxic substances. Animal studies: Compared to controls, rats (both male and female), male mice, and hamsters that ingested BHA had prolonged lifespans and developed benign and malignant tumors (papilloma and squamous cell carcinoma) in their forestomach. Rats were able to tolerate up to 1200 ppm of BHA in their diet for up to 21 months. No histopathological or carcinogenic effects associated with BHA intake were observed, nor were any effects on reproduction found. Teratogenic effects of BHA were assessed in rats and mice at dose levels up to 1000 mg/kg and 500 mg/kg, respectively. No abnormal results related to exposure were observed. In another study, the teratogenic effects of BHA in rabbits were assessed at dose levels ranging from 50 to 400 mg/kg. No abnormal results were reported. Feeding BHA to juvenile fish (hermaphroditic killifish) led to hepatocellular carcinoma in adult fish. In mice, doses of BHA ranging from 5 to 500 mg/kg increased serotonin utilization in the central nervous system, resulting in neurological alterations. Ecotoxicity studies: Dietary BHA altered the activity of mixed-function oxidase systems in rainbow trout liver microsomes, changed ethyl isocyanate binding rates, and decreased cytochrome P-450 levels.

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Non-human toxicity values
Oral LD50 in rats: 4000 mg/kg
Oral LD50 in rabbits: 2100 mg/kg
Oral LD50 in mice: 2000 mg/kg body weight
Interactions
In the benzo[a]pyrene-induced ICR/HA forestomach tumor assay in mice, the addition of 3-tert-Butylated-4-hydroxyanisole reduced the number and incidence of tumors.
Mice fed a diet containing 0.75% 2(3)-tert-Butylated-4-hydroxyanisole (BHA) for 8 days showed a 50% reduction in the maximum induction of ornithine decarboxylase (ODC) after BHA supplementation. Mice treated with TPA (12-O-tetradecanoylphorbol-13-acetate) showed a significant inhibitory effect compared with mice fed a control diet. Topical application of BHA (55 μmol) 30 minutes before treatment with TPA (17 nmol) inhibited promoter-induced ODC activity by up to 80%. BHA administration 16 hours before or 2 hours after promoter treatment had no effect. The inhibitory effect was dose-dependent, with a dose of 6 μmol inhibiting 50% of ODC-induced activity. Structure-activity studies showed that the hydroxyl and tert-Butylated substituents were key determinants of inhibitory activity. Rats were fed a diet with or without ciprofibrate (10 mg/kg body weight) supplemented with 2(3)-tert-Butylated-4-hydroxyanisole (0.5% wt/wt) for 60 weeks. 2(3)-tert-Butylated-4-hydroxyanisole significantly reduced the incidence and number of hepatocellular carcinomas larger than 5 mm. The data suggest that the inhibitory effect of BHA on ciprofibrate-induced liver tumorigenesis may be attributed to its H₂O₂ and free radical scavenging properties, as it does not inhibit peroxisome proliferation or H₂O₂ peroxisome induction in the livers of ciprofibrate-fed rats. In mice and rats, the LD50 of BHA was determined by intraperitoneal or oral administration, using dimethyl sulfoxide (DMSO) or olive oil as solvents. When using DMSO, the LD50 of intraperitoneal BHA was approximately two orders of magnitude lower than the oral LD50. This difference was not significant when BHA was dissolved in olive oil. BHA and BHT both exhibit chemopreventive effects when co-administered with various carcinogens affecting different target organs. Antioxidants enhance the scavenging capacity of detoxification enzymes against carcinogens, and many antioxidants also scavenge free radicals. The structures of these substances suggest they are unlikely to be electrophilic, and genotoxicity tests were all negative. Both BHA and BHT have been shown to inhibit intercellular communication in cultured cells. Drinking water disinfection byproducts (DBPs), generated during water chemical disinfection, may pose a threat to public health. Two main types of DBPs are present in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed after chlorination, where chlorine reacts with iodides and bromides in water. Previous studies have shown that HAAs possess cytotoxic, genotoxic, mutagenic, teratogenic, and carcinogenic properties. This study aimed to determine the effects of haloacetic acids (HAAs) on human somatic and germ cells, and whether oxidative stress is involved in their genotoxic effects. This study examined peripheral blood lymphocytes and sperm cells, among other somatic and germ cells. The effects of three HAA compounds—iodoacetic acid (IAA), bromoacetic acid (BAA), and chloroacetic acid (CAA)—were investigated. After determining appropriate concentrations, the scavenging effects of the antioxidant Butylatedated hydroxyanisole (BHA) and catalase on oxygen free radicals were studied under alkaline conditions (pH > 13) using single-cell gel electrophoresis (comet assay) and micronucleus assays. The comet assay results showed that both BHA and catalase reduced DNA damage in all cell types compared to HAAs alone. In the micronucleus assay, micronuclei (MNis) were found in peripheral blood lymphocytes exposed to all three glycolic acid copolymers (HAAs). Catalase and Butylatedated hydroxyanisole (BHA) generally reduced the induction of MNis, indicating that oxygen free radicals played a role in both assays. These observations raise public health concerns due to the similar genotoxic responses observed in both human somatic and germ cells. Butylatedated hydroxyanisole and propylparaben are commonly used phenolic preservatives in food, pharmaceutical, and personal care products. Both chemicals have undergone extensive toxicological studies due to growing concerns about their potential environmental and human health impacts. However, the cytotoxicity and underlying mechanisms of co-exposure to these compounds have not been explored. This study analyzed a range of relevant cytotoxic endpoints, including cell viability and proliferation, oxidative stress, DNA damage, and changes in gene expression, to assess whether the antioxidant Butylatedated hydroxyanisole could prevent the pro-oxidative effects induced by propylparaben in Vero cells. We demonstrated that the binary mixture of the two chemicals produced a stronger cytotoxic effect than exposure to each compound alone. Co-treatment with Butylatedated hydroxyanisole (BHA) and propylparaben induced G0/G1 phase cell cycle arrest due to enhanced oxidative stress and DNA double-strand break generation. DNA microarray analysis revealed that the interaction between transforming growth factor β (TGFβ) and ataxia-telangiectasia mutant kinase (ATM) pathways modulates the response of Vero cells to the test compounds in the binary mixture. Our results indicate that BHA enhances the pro-oxidative effects of propylparaben in cultured mammalian cells and provide useful information for assessing its safety. We evaluated the anti-androgenic effects of Butylatedparaben (BuPB), BHA, BHT, and propyl gallate (PG) individually and in combination (binary mixture) using the MDA-kb2 cell line. Exposure of these cells to androgen receptor (AR) agonists induced the expression of a reporter gene (encoding luciferase), and the activity of the reporter protein was monitored by measuring luminescence intensity. In evaluating anti-androgenic effects, we tested individual test compounds or binary mixtures in the presence of a fixed concentration of a potent AR agonist (1000 pM 5α-dihydrotestosterone; DHT). Cell viability was assessed using a resazurite-based assay. This is the first report in the literature of the (anti)androgenic activity of PG. Neither the individual compounds nor the binary mixtures showed androgenic activity. BuPB, BHA, and BHT all exhibited weak anti-androgenic activity in the presence of DHT, which was also confirmed in the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT, and BHA+BHT). In addition to in vitro testing of the binary mixtures, the accuracy of two mathematical models (dose-additive and response-additive models) in predicting the anti-androgenic effects of the selected binary mixtures was evaluated. The dose-additive model ensured a good correlation between experimental and predicted data. However, since compound PG did not show any effect in the individual tests, mixtures containing PG could not be evaluated. This study aimed to investigate the protective effect of Butylatedated hydroxyanisole (BHA), a commonly used phenolic antioxidant in food, against ferric triacetate (Fe-NTA)-induced nephrotoxicity. Four- to six-week-old male albino Wistar rats weighing 125-150 g were used. After treatment with BHA (1 and 2 mg/rat/day), the animals were given a single dose of Fe-NTA (9 mg/kg body weight). Compared with the saline control group, Fe-NTA treatment increased ornithine decarboxylase (ODC) activity in the kidneys by 5.3-fold, and [(3)H]-thymidine incorporation into DNA by 2.5-fold, while glutathione (GSH) levels and antioxidant enzyme activities decreased by 2-2.5-fold. BHA pretreatment significantly reversed these changes. Compared with the Fe-NTA treatment group, at a BHA dose of 2 mg/day/rat, ODC activity and DNA synthesis decreased by 2.12-fold and 1.15-fold, respectively. Pretreatment with BHA before Fe-NTA treatment increased GSH levels and antioxidant enzyme activity in the kidneys by 1.5–2 times. The results indicated that BHA could inhibit Fe-NTA-induced nephrotoxicity in male Wistar rats. For more complete data on interactions of Butylatedated hydroxyanisoles (32 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 4000 mg/kg
Oral LD50 in rabbits: 2100 mg/kg
Oral LD50 in mice: 2000 mg/kg body weight
Oral LD50 in rats: 2200 mg/kg body weight
Intraperitoneal LD50 in male rats: 881 mg/kg body weight
In primary rat/mouse astrocytes, Butylatedated hydroxyanisole (BHA) (≥25 μM, 24 h) induces endoplasmic reticulum stress-mediated apoptosis, characterized by CHOP upregulation, caspase-3 activation and increased number of apoptotic cells. Percentage [1][2]
In C57BL/6 mice fed 0.1%–0.4% Butylatedated hydroxyanisole (BHA) for 14 days, no significant hepatotoxicity (no change in serum ALT/AST levels) or intestinal mucosal damage (histological examination) was observed. Histological examination revealed mild vacuolation of renal tubular cells [3]

[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 (BHA) is a white, beige, or slightly yellow waxy solid with an aromatic odor and a slightly bitter, burning taste. (NTP, 1992)
3-tert-Butylated-4-hydroxyanisole is an aromatic ether, a derivative of 4-methoxyphenol, in which a hydrogen atom at the ortho position of the phenolic hydroxyl group is replaced by a tert-Butylated group. It has antioxidant properties and is also a product of human xenobiotic metabolism. It belongs to the phenolic and aromatic ether compounds.
2-tert-Butylated-4-methoxyphenol has been reported to exist in Salvia officinalis, Murraya paniculata, and Dillenia indica, and there is relevant data.
Mechanism of Action
Administration of 2(3)-tert-Butylated-4-hydroxyanisole (BHA) to rodents can protect various target tissues from tumorigenesis induced by various chemical carcinogens. BHA can reduce the levels of benzo[a]pyrene and mutagenic metabolites produced by various therapeutic drugs in vivo; it can increase the activity of hepatic microsomal epoxide hydratase and cytosolic glutathione S-transferase; it can alter the activity of other liver enzymes and affect the levels of certain liver catalytic components; it can also increase the concentration of non-protein thiols in the liver and several other tissues. /BHA/
This study investigated the effects of Butylatedated hydroxyanisole (BHA) on the activity of aryl hydrocarbon hydroxylase (AHH) in the liver, lungs, and skin of rats and mice to study its possible anticancer mechanism. This study used AHH inducers, 3-methylcholanthrene, phenobarbital, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Observations revealed that commercially available mixtures of 2-tert-Butylated-4-hydroxyanisole, 3-tert-Butylated-4-hydroxyanisole, and BHA (85% 3-BHA and 15% 2-BHA) exhibited roughly the same inhibitory efficacy against microsomal AHH, although the 2-isomer appeared to have a slightly stronger inhibitory effect. Data suggest that the inhibitory effect observed in commercial BHA is a result of the combined action of both isomers and is dependent on animal species, tissue type, and inducer treatment. Both BHA and BHT exhibit chemopreventive effects when co-administered with various carcinogens affecting different target organs. Antioxidants can increase the detoxification enzymes of carcinogens, and many antioxidants also act as free radical scavengers. The structures of these substances suggest they are unlikely to be electrophilic, and genotoxicity tests were all negative. Both BHA and BHT have been shown to inhibit intercellular communication in cultured cells. Estrogen metabolism-mediated oxidative stress is believed to play an important role in estrogen-induced breast cancer development. We have previously demonstrated that the antioxidants vitamin C (Vit C) and Butylatedated hydroxyanisole (BHA) can inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and suppress breast cancer development in August Copenhagen Irish (ACI) female rats. This study aimed to elucidate the mechanism by which these antioxidants prevent DNA damage during breast cancer development. We treated ACI female rats with E2, Vit C, Vit C+E2, BHA, and BHA+E2 for up to 240 days. In the mammary tissue and breast tumor tissue of the E2-treated rats, we quantitatively analyzed the mRNA and protein levels of the DNA repair enzyme 8-oxoguanine DNA glycosidase (OGG1) and the transcription factor NRF2, and compared these levels with those in the antioxidant-only or antioxidant-in-combination-E2 treatment groups. OGG1 expression was suppressed in the estradiol (E2)-treated rat mammary tissue and breast tumors. NRF2 expression was also significantly suppressed in E2-treated breast tissue and breast tumors. Vitamin C or BHA treatment prevented the E2-mediated reduction of OGG1 and NRF2 levels in breast tissue. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated OGG1 induction was achieved by increasing the direct binding of NRF2 to the OGG1 promoter region. Studies using silent RNA confirmed the role of OGG1 in inhibiting oxidative DNA damage. Our study suggests that the antioxidants vitamin C and BHA provide protection against oxidative DNA damage and E2-induced breast cancer at least in part through NRF2-mediated OGG1 induction. Butylatedated hydroxyanisole (BHA) is a synthetic phenolic antioxidant widely used as a food additive to prevent lipid peroxidation [1][2][3]. Its neurotoxic mechanism in astrocytes involves two key pathways: cytoplasmic calcium overload (possibly through endoplasmic reticulum calcium release) and subsequent endoplasmic reticulum stress, leading to apoptosis. Endoplasmic reticulum stress inhibitors or calcium chelators can alleviate this toxicity [1][2]
In mouse liver, Butylatedated hydroxyanisole (BHA) activates the Nrf2 signaling pathway to induce detoxification enzymes, while its effect on intestinal Nrf2 is negligible, suggesting that there is tissue-specific regulation of xenobiotic metabolism [3]
The literature [4] mainly focuses on the mechanism of ferroptosis and does not include data related to Butylatedated hydroxyanisole (BHA) [4]

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.

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