TOXICOLOGICAL POTENTIAL OF PARABENS- A WIDELY USED PRESERVATIVE

TOXICOLOGICAL POTENTIAL OF PARABENS- A WIDELY USED PRESERVATIVE SHRUTI GOYAL, SAROJ KUMAR AMAR, HARI NARAYAN KUSHWAHA, JYOTI SINGH, AJEET KUMAR SRIVASTAV, DIVYA DUBEY, DEEPTI CHOPRA, R.S. RAY * Photobiology Division, CSIR- Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, ABSTRACT Parabens are commonly used as antimicrobial preservatives in a variety of personal care, pharmaceutical, cosmetics, and food products. It is a group of synthetic chemicals that person exposed every day. Parabens are ubiquitously present in waste water, river, soil and house dust. It has also been detected in human tissue and body fluids. It also acts as endocrine disrupter at high exposure levels. Studies in young male rats exposed during development have shown adverse effects on sperm production and testosterone levels following exposure to parabens with longer side chains i.e. butyl, -isobutyl and propylparabens. After dermal uptake, parabens are hydrolyzed, conjugated with protein and excreted in urine. Despite high total dermal uptake of parabens and its metabolites, little intact parabens can be recovered in blood and urine. Overall the estrogenic parabens and their metabolites in blood may exceed the action of endogenous estradiol. The interaction between parabens and mitochondrial function in the testis may be key in explaining the contribution of parabens for a decrease in reproductive potential. Thus Parabens which has been considered as a preservative in cosmetic may have harmful effects on Human skin. So it is important to assess the toxicological potential of parabens. Keywords: Parabens, endocrine disruption, estrogen, testosterone, skin. Introduction Parabens are widely used as preservatives in food, cosmetics and pharmaceuticals products. It is a group homologous series of hydroxylbenzoic acid, esterifies at the C-4 position (including methyl-, ethyl-, propyl-, butyl-, benzyl- and heptyl-, parabens), used singly or in combination to exert an antimicrobial effect. Parabens were first used as antimicrobial preservatives in pharmaceutical products in the mid- 1920s (1Sabalitschka, 1930). Parabens have proved to be very effective antimicrobial agents and are widely used. As the chain length of the ester group of paraben increases, antimicrobial activity increases, but water solubility decreases ( Elder, 1984). Parabens occur naturally in foods. Methyl paraben has been reported as a ingredient of cloudberry, fruit juice, white wine and Bourbon vanilla, and recently, propyl paraben 77

has been detected in the aerial part of the plant Stocksia brahuica (Family: Sapindaceae) (Ali et al., 1998). In 1974, JECFA (Joint FAO/WHO expert committee on Food additive) recommended the Acceptable Daily Intake (ADI) for methyl, ethyl, propyl ester of p hydroxybenzoic acid as 0-10 mg/kg body weight/ day. Figure 1 shows the parabens family which are actively used as preservatives. 1.1. Economic uses 1.1.1. Use as a food ingredients Parabens are employed in several foods including processed vegetables, baked goods, fats and oils, sugar, coffee extracts, fruit juices, sauces, pickles,soft drinks, pudding and frozen dairy products at concentrations of between 450 and 2000 ppm ( Daniel, 1986). Among the parabens, methyl and propyl parabens are the most extensively present in food. Parabens is also used as antimycotic in food packaging materials with no limit or restriction. 1.1.2. Use in cosmetics Parabens are widely used as preservatives in regular based cosmetics products including face, body creams, Lotions and moisturizers, eye makeup products, foundation, shampoo, deodorants etc. Methyl and propyl parabens are the major that are used in cosmetics. Rastogi et al. investigated the contents of parabens in cosmetic products and found a preferential use with decreasing order of methyl- > ethyl- > propyl- > butyl- > benzylparaben in various cosmetic products. The use of parabens in cosmetic products upto a maximum conc. of 0.8% (w/w) is permitted by the Danish and EEC regulations ( Howlett, 1992; Rastogi et. al., 1995). 1.1.3. Pharmaceutical uses Parabens were first used as a preservative in pharmaceutical products. Among the parabens, propyl paraben is one of the most effective fungistats used in pharmaceutical preparations. A variety of drug formulations including suppositories, anesthetics, pills, syrups, weight gaining solutions and injectable solutions are known to contain parabens as preservatives. Methyl parabens and Propyl parabens are used in a number of over the counter (OTC) drugs. The concentration of parabens is varies from product to product but seldom exceeds 1%. Recent reports have indicated that exposure to parabens may modulate or disrupt the endocrine system and thus may have harmful consequences on human health. These studies have indicated that parabens possess weak estrogenic activity, these also an association between the use of underarm cosmetics containing parabens and an increased incidence of breast cancer. Additionally, some recent studies have reported adverse effect of parabens on reproductive system. Energy metabolism is the fundamental process supporting all cell functions 78

and is crucially important in sperm, which are specialized motile cells that have to move rapidly to encounter and fertilize the oocyte. To this regard sperm need unusually more ATP than any other cell. Inhibitory effects of parabens on mitochondrial respiratory capacities have been known since 1998. Martins et al. have also demonstrated that parabens inhibit respiration and depress mitochondrial membrane potential on isolated testis mitochondria in a side chain length- and concentration-dependent manner. The purpose of this review is to evaluate the toxic effect of parabens on human beings. 1.1.4. Mode of Action The definite cause and effect as to how parabens work has not been established yet, but studies suggest that parabens may be most active at the cytoplasmic membrane level. In the presence of parabens, Serratia marcescens caused leakage of cellular RNA, which was detected by Furr and Russell in 1994. It was found that amount of leakage and alkyl chain length of the parabens was proportional to each other. In 1973, Freese et al gave the conclusion that the parabens were capable of inhibiting both membrane transport and ETS when they found that parabens can not only inhibit serine uptake but oxidation of α-glycerol phosphate and NADH in membrane vesicles of Bacills subtilis too (Freese et al., 1973). Due to the fact that parabens cause leakage of cellular contents, in 1980, Eklund postulated that they are capable of neutralizing chemical and electrical forces that establish a normal membrane gradient. In the year 1960, Oka suggested that instead of the cell fluid or lipid layers, parabens act on yeast by absorbing on the solid phase of cells. Valkova et al studied the resistance of bacteria to the parabens in 2002. Report showed that a strain of Enterobacter cloacae produced an esterase that was responsible for the hydrolysis of parabens. In some bacterial species, they are postulated to act by disrupting membrane transport processes or by inhibiting synthesis of DNA and RNA or of some key enzymes, such as ATPases and phosphotransferases, therefore, parabens are prominent antimicrobial agents in a variety of industries. 2. Current status of research and development The recent research claims that the levels of some parabens were higher in breast tissue from the women who had used underarm cosmetics compared with those who had never used or no longer used underarm cosmetics. Now scientists have found evidence that these chemicals might be contributing to the obesity epidemic. Further research has shown that parabens may affect other hormonal systems in the body, placing them in a group of chemicals called endocrine disruptors. Scientists have suspected that some endocrine disruptors might affect the 79

body s metabolism. As these compounds build up in the environment, it could be contributing to the current obesity epidemic, parabens could also act as a thyroid hormone receptor agonist/antagonist and as an agonist for peroxisome proliferator activated receptor (PPAR) (Taxvig et al., 2012). Together, these studies suggest that parabens are endocrine disrupting compounds (EDC). Recent studies have suggested that parabens are endocrine disruptors along with estrogenic, antiandrogenic activities and also shown adipogenic effect (Pan Hu et.al., 2013). A number of current studies have established potentials of parabens into human body in intact and unmetabolized forms. It has been found that parabens are quickly absorbed in GI or skin followed by Phase I and Phase II enzymatic reaction earlier than being excreted from the urine, which to some extent contributes to their extensive inertness and low toxicity (Darbre, 2008). Several European studies have shown that the occurrence of parabens in semen, milk samples, blood and urine in the population of Europe. It was also assumed that systemically absorbed low concentration of parabens might accumulate in tissue of body and consequently it diffusion to the breast region (Darbre 2008,2004). Though, this clinical studies did not ascertain a obvious association between expansion of breast cancer and paraben.in 2014 Khanna S et.al., was first time investigated that in In vitro, parabens can affect migratory and persistent attribute along with proliferation of human breast cancer cell lines MCF-7, T-47-D, ZR-75-1(Khanna S et.al., 2014). In some report it has been shown that possibly Parabens also contribute to the Obesity by affecting the metabolism of lipid or adipogensis. (Pan Hu et.al., 2013).In one report it has been shown that concentration of paraben in urine of 554 pragnent women a pilot study done by National Children's Study,there was a significant racial difference was found (Mortensen ME1,2014).Paraben can cause autism attribute during combine prenatal and postnatal exposure it was observed that neurodevelopmental disorders was similar to some extent of neurological disorders VA model of autism.( Ali EH et.al., 2013) Low-dose UVB irradiation alone and practical concentrations of MP (methyl paraben) alone did not induce cell death in HaCaT keratinocytes. Moreover, MP or UVB alone induced little or no ROS and NO production and NFkB and AP-1 activation in HaCaT keratinocytes. However, practical concentrations of MP significantly enhanced ROS and NO production, NFkB and AP-1 activation, and apoptosis of UVB exposed HaCaT keratinocytes. Because ROS and NO production has been reported to modulate transcription factors (Handa et al., 2004), UVB irradiation of MP- 80

treated HaCaT keratinocytes may activate these transcription factors via ROS and NO production, thus inducing production of various cytokines and chemokines. This, in turn, could result in a vicious circle of inflammation, ultimately resulting in cell death. 3. Parabens Toxicity 3.1. As an endocrine disrupting agent: In general, estrogen plays a central role in the growth, progression and treatment of breast cancer (Miller, 1996; Lonning, 2004). All the widely used parabens have been shown to possess estrogenic activity to different extent in different assay systems in vitro and in vivo. The estrogenic activity of parabens is known to increase with increasing chain length and with branching of the alkyl chain (Byford et al., 2002; Routledge et al., 1998). However, in relation to estrogen as a control during in vivo studies, the estrogenic activity of parabens is not as prominent as estrogen. Parabens binds to estrogen receptor stimulating ER-dependent cell growth and influencing the expression of estrogen-dependent genes.while exposure to sufficient doses of exogenous estrogen can increase the risk of certain adverse effects. However, recent studies have now shown that some xenestrogens may be able to exert endocrine disrupting properties by interfering with metabolic enzymes responsible for the synthesis of physiological estrogens or for modification of their availability in free, unconjugated form (Whitehead et al., 2006). Prusakiewicz et al. report that parabens are able to inhibit sulfotransferases. The finding that parabens can also inhibit sulfation of estrogens through inhibition of SULTs suggests that parabens may also indirectly enhance estrogen effects through elevation of free estradiol levels (Prusakiewicz et al., 2006). The potency of SULT inhibition increased as the ester chain length increased. Figure 2 show parabens interaction with the estrogen sulfation cycle in skin. 3.2. Cause of breast cancer As estrogen is a major etiological factor in the growth and development of the majority of cases of human breast cancer. The use of parabens in cosmetics, particularly underarm deodorants and antiperspirants, may contribute to the rising incidence of breast cancer. A clinical observation shows a disproportionately high incidence of breast cancer in the upper outer quadrant of the breast, to which these cosmetics are applied. Recent results of the presence of paraben in tumor tissue (Darbre et al., 2004) along with results of the earlier described studies on the estrogenic potential of parabens lead to the hypothesis that parabens may be involved or contribute to the incidence of breast cancer. In a recent study, parabens were detected in a small number (n = 20) of tissue samples from human breast 81

tumors (Darbre et al., 2004). Of the parabens detected, methyl paraben represented approximately 60% of the total parabens. Other parabens detected were ethyl-, n-propyl-, n-butyl- and isobutyl-paraben. 3.3. Effect on male reproductive system Recent studies reported adverse reproductive effects of parabens. Although there is increasing concern regarding the effects of parabens, as mentioned previously, a possible role on the increased incidence of breast cancer (Darbre et al., 2004), little data exists on their effects regarding human sperm, although a number of studies with animal models were performed. Rodent exposure to butylparaben (Oishi et al., 2001 & 2002) and propylparaben adversely affected testosterone synthesis and male reproductive function. Also, and more importantly, male reproductive organ weights (testes, seminal vesicles and prostate glands), sperm counts and sperm motility were adversely affected as well as the number of round and elongated spermatids at stage VII of the seminiferous tubule after exposure to parabens. Vaginal opening also occurred earlier in female offspring, compared with the control group (Dhooge et al., 2006). Oishi reported no treatment-related effects of propylparaben on reproductive organ weights but decreased epididymes and seminal vesicles weights after dietary butylparaben intake were recorded. Furthermore, a significant decline of caudal epididymal sperm reserves was recorded for methyl- and butylparaben consumption. Moreover, sperm concentration decreased in a dose-dependent manner and the same was observed with daily sperm production (DSP) and efficiency of sperm production in testes (DSP/g testes). Inhibitory effects of parabens on mitochondrial respiratory capacities have been known since 1998 (Nakagawa et al., 1998). Martins et al. have also demonstrated that parabens inhibit respiration and reduce mitochondrial membrane potential on isolated testis mitochondria in a side chain lengthand concentration-dependent manner (Martins et al., 2008). It was demonstrated that butyl- and isobutylparaben were more toxic than propyl- and isopropylparaben, and ethyl- methylparaben and p- hydroxybenzoic acid were less toxic than propylparaben, when considering mitochondria as the site for the toxic effect (Nakagawa et al., 1998). Later, it was pointed out that the MPT pore is involved in the toxicity of different parabens in hepatocytes and isolated liver mitochondria (Rylova et al., 2007). The results are extremely important as they are first to demonstrate that the paraben-induced uncoupling and decrease of ATP synthesis is associated with MPT induction. 82

3.4. Early onset of puberty Another correlation that has been found regarding parabens is the relationships between their usage and early onset of puberty in young girls and boys. Parabens mimic naturally occurring hormones like estrogen and testosterone, and interfere with many hormonal pathways. They have the potential to induce as earlier than usual onset of puberty- a growing trend that is becoming more prevalent nowadays. For instance, endocrinologists have noticed that the average age of puberty has decreased in recent decades, with girls as young as eight years old developing breasts and young boys experiencing testicular enlargement and pubic hair growth earlier than ever before. 3.5. Role in early aging Several commercial products that contain parabens are beauty products are supposed to enhance the skin, such as moisturizers and lotions; research has found that they accelerate the skin aging process. Researchers from the Kyoto Prefectural University of Medicine in Japan found that the methylparaben increases sensitivity to skin that is, when exposed to ultraviolet radiation, skin cells die more rapidly than normal. These UV rays causes DNA damage and can lead to photo-aging and skin damage such as wrinkles and premature aging caused by excessive free radical damage. Figure 3 shows parabens interaction with several molecular pathways within the skin. 4. Conclusions and Perspectives Parabens exposure comes mainly from the use of personal care products containing these compounds. While their exact health effects are currently unknown, they do possess some estrogenic activity, can adversely affect the breakdown of endogenous estrogens, and cause mitochondrial dysfunction. These compounds cause breast cancer, male infertility and early aging also. With widespread presence of parabens in urines across the population, there is a need to equally understand distribution of parabens in all body tissues and beyond breast, to now investigate the distribution of parabens across all endocrine-sensitive tissues which might be influenced through topical exposure to the parabens, not least male reproductive organs in the early year of life and skin itself. Future research should definitely be done on the different types of parabens containing different alkyl R groups. For example, a past experiment showed that methyl paraben is able to cause pain sensation through particular ion channels (Fujita, 2007). Improved studies on absorption, distribution, metabolism and elimination (ADME) of Parabens are required. In humans, improved studies on uptake and metabolism of parabens in human as well as background populations are needed. An extended one generation study or a two generation study on 83

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