Oxidative stress: Insights into the Pathogenesis and Treatment of Alopecia

Shweta Ramkar, Hemendra Kumar Sahu, Narayan Hemnani, Ravi Parashar, Preeti K. Suresh*

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India.

 Abstract:

Hairs are exposed to a host of endogenous and environmental stress by pollutants, microbial assaults, UV radiation, oxidized scalp lipids, grooming practices and cosmetic treatments which have diverse range of adverse consequences. The exposure to these environmental and cosmetic substances, leads to generation of free radicals, reactive oxygen species in particular, leading to oxidative stress. Oxidative stress generates inflammation, and/or psycho-emotional stress, and also influences the ageing process, including the hair follicle. The term alopecia signifies loss of hair owing to several factors, ultimately resulting in decreased hair density. Cell death on hair follicle (keratinocytes and its distinctive mesenchyme of dermal papilla) have been attributed to mechanisms of oxidative stress, including H₂O₂, nitric oxide and derivatives, ultraviolet rays, ionizing radiations, endotoxin-induced inflammation, photodynamic therapy and cigarette smoke. Persistent oxidative activities in the body, may generate antioxidant defense systems, which can prevent the attack of biological molecules. In case of androgenic alopecia, copper and zinc was discovered in the disrupted metabolism form in serum, urine and hair of the patients, and data suggests rise in oxidative stress. This review is focused on the effects of the reactive oxygenated species in disturbing the redox balance and inducing oxidative injury that leads to androgenic alopecia.

Keywords: Hair Follicle, Oxidative Stress, Antioxidants, Reactive Oxygen Species, Redox Balance.

1.   INTRODUCTION

Oxidative stress is caused by the imbalance between the production of free radicals and ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants, leading to tissue damage (Haslam et al., 2016). Free radicals are the atom or the group of atoms that contain unpaired electron and are formed during a number of biochemical reactions (Suresh et al., 2013). They are highly reactive and may cause damage to macromolecules like DNA, protein, lipid, enzymes, etc. They are also known as reactive oxygen species (ROS). Antioxidants are one of the defensive strategies that assist in controlling the level of free radical and prevent the oxidation of biomolecules. Oxidative stress have been implicated in a number of human pathologies including neuro-degenerative disorders, chronic kidney disease, skin aging and common dermatoses such as psoriasis (Richter et al., 2015; Wagener et al., 2013; Haslam et al., 2016, Suresh et al., 2014). Oxidative stress is reported to be one of the key influences leading to hair loss as it is linked with a number of factors that increase cellular oxidative stress, including smoking, alcohol consumption, some metabolic syndrome, and UV radiation (Upton et al., 2015; Gao and Dalton, 2007). Some of the common examples of enzymatic antioxidants in alopecia include superoxide dismutase, catalase_, glutathione peroxidase_, paroxone, nemoxigenase and thioredoxin reductase. Non-enzymatic antioxidants include glutathione, vitamin E and beta-carotene. In excess amount, the antioxidants may damage protein structure (Yasuko et al. 1994). The reaction of human organs on oxidative damage and how they control redox insult is of significant physiological and clinical relevance.

2. ALOPECIA

Androgenic alopecia (AGA) is a common hair loss dysfunction and is also referred to as hereditary/genetic hair loss. Accordingly, there are two conditions viz., male pattern baldness (MPB) and female pattern baldness (FPB). It may occur as early as in the late teens or occasionally in early twenties. The phenotypical changes are produced when 5-alpha reductase enzyme starts transforming the testosterone hormone into its derivative, dihydrotestosterone (DHT) which induces the miniaturization of the hair follicles and the hair cycle changes, and ultimately hair no more extrudes through the skin ( Prie et al., 2016). Mantel et. al. (2017) reported that substantial levels of prostaglandin-D2 was present in the bald scalp of AGA patients and it functionally inhibits the hair growth (Mantel et al., 2017). Alopecia areata (AA) is an autoimmune disorder with body's own immune system assaulting the healthy hair follicles leading to patchy hair loss on the scalp. The actual explanation for generation of AA remains unclear, however it is commonly assumed to be induced by stress and stressful events. In certain situations, AA can advance throughout the entire scalp, and this is termed as alopecia totalis (AT). In alopecia universalis (AU), significant hair loss occurs across the entire body, including eyebrows (Islam et al., 2015). In cicatricial alopecia (CA) or scarring alopecia hair follicles are damaged and replaced by scar tissue. In case of primary CA, hair loss occurs owing to direct inflammation of the hair follicles. Secondary CA usually refers to scarring hair loss (such as burns or infections), and ensues as a result of an event or process unrelated to the follicles. Traction alopecia (TA) is a distinct kind of alopecia and is frequently produced directly by the action of the individual, which results in excessive stress on the hair and breakdown (Price and Colombe, 1996). It may be caused by traumatic hair styles (e.g., braiding, tight ponytails and frequent hair treatment with chemicals (e.g., hair coloring and bleaching). Exposure to allergens, irritants, toxins, burns, injuries, and infection as well as certain medication (especially antibiotics, testosterone), chronic kidney failure, radiation and chemotherapy and malnutrition have also been attributed to the generation of alopecia (Ramkar et al., 2020).

3. ANTIOXIDANT AND ITS ACTION

Antioxidants are the substances that prevent or inhibit the factors that lead to oxidative injury in the cell. When an antioxidant reacts with a free radical, antioxidant itself becomes oxidized. Therefore, the antioxidant resources must be regularly stored in the body. Antioxidants perform diverse roles in different systems, for instance an antioxidant effective against free radicals in one particular system may be ineffective in other systems. Also, in certain situations, these antioxidants may even act as a pro-oxidant as it can generate toxic ROS/RNS (Abdel et al., 2011). The antioxidant process can function in one of the two ways, viz., it can break or prevent the chain reaction or assist in disintegrating into a harmless product. In the chain-breaking process, when a radical is released or steals an electron, a second radical is formed. In this continuous process, the last one exerts the same action on another molecule until the free radical does not form a stable compound by a chain-breaking antioxidant, or it is simply converted into an innocuous product (Ryter et al., 2007). The classic example of such a chain reaction is lipid peroxidation. An antioxidant enzyme like superoxide dismutase, catalase and glutathione peroxidase can prevent oxidation by reducing the rate of chain initiation, e.g., either by scavenging initiating free radicals or by stabilizing transition metal radicals such as copper and iron (Akar et al., 2002).

4. OXIDATIVE STRESS AND PATHOPHYSIOLOGY OF ALOPECIA

Populations of pluripotent stem cells are responsible for regulation of hair follicle development as well as hair growth and are present in dermal papilla (DP). In DP cells (DPCs), attachment of androgen in androgenic receptors is believed to be action on hair growth regulation. Studies indicate that in greying follicles, ROS was produced by the melanocytes and it might be a reason for creating an oxidative environment in the surrounding follicular area that could affect DPCs especially in balding scalp (Driskell et al., 2013; Hunt and McHale, 2005). As depicted in Fig.1 free radical causes the oxidative damage to hair follicle stem cells or dermal papilla cells in epithelial bulge, which are responsible for the repetitive growth of hair follicle during hair cycle (Arck et al., 2006).

Fig. 1: Sequential events in pathogenesis of alopecia

Ultraviolet (mainly UV-B) radiation attacks the keratin (hair protein fractions) and melanin pigments. On the surface of the cuticle and inside the hair fibre, disulfide bonds are present that are broken by UV-B radiation leading to denaturation of the hair structure. On the other hand, UV-A radiation generally produces ROS via interaction with endogenous photo sensitizers (Fernández et al. 2012). Fig. 2 demonstrates the cellular damage in hair follicle.

Fig. 2: Schematic illustration of the UV-induced alopecia

Alopecia may be a fall out of radiation therapy, chemotherapy, stem cell transplantation, and targeted therapy. These therapies can cause hair loss by damaging the cells that promote hair growth. Hair loss may occur throughout the body, including the head, face, arms, legs, underarms, and pubic area (Ramkar et al.,2022). Hair may come out entirely, slowly, or in parts. Or, hair will simply become thin, sometimes unnoticeably and may become duller or dryer. Hair loss associated with cancer therapy is usually transitory. Most of the time, hair will grow back, however infrequently, it may remain thin (Gilhar et al., 2016; Harman, 2002). Some of the drugs likely to cause hair loss or thinning are listed in Table 1.

Table 1- Drugs with oxidant properties

5. NUTRIENT ANTIOXIDANTS

The nutrient antioxidant deficiency is one of the major factors, which can cause numerous chronic and degenerative pathologies. Hair growth can be maintained by the combination of nutrients delivered to the hair roots and good health. It is also affected by the androgens enzyme blockers without the possibility of side effects from the use of hair loss medications ( Sinbad et al., 2019). Right choice of antioxidants, minerals, vitamins, and amino acids may promote hair growth. However, excess use of antioxidants, vitamin A, vitamin E, omega 3 can cause hair loss instead of hair growth (Rutkowski and Grzegorczyk, 2012; Timbo et al., 2006; Rajput, 2017). There are number of nutrients present in the body that have unique structure and antioxidant function (Radimer et al., 2004).

Table 2- Mechanism of action of various nutrients

Vitamin E

Vitamin E is a fat-soluble vitamin with high antioxidant potency. It is a chiral compound with eight stereoisomers: α, β, γ, δ tocopherol and α, β, γ, δ tocotrienol. In humans, only α-tocopherol is the bioactive form. α-tocopherol safeguards the membranes around the cell from damage by free radicals owing to its fat-soluble properties (Heyland et al., 2005; Sinbad et al., 2019). Studies have indicated that vitamin E may have preventive action against breast, prostate and colon cancers, ischemia, some cardiovascular diseases, arthritis, cataract and certain neurological disorders. Cooking and storage may destroy the natural d-α-tocopherol in foods (Devasagayam et al., 2004).

Vitamin C

Vitamin C or ascorbic acid is a water-soluble vitamin, and have been implicated in the formation of carnitine collagen and neurotransmitters biosynthesis. They are antioxidant, anti-atherogenic, anti-carcinogenic and immunomodulator (Yadav et al., 2016). It has also demonstrated reduction in the incidence of stomach cancer, colorectal and lung cancer. Vitamin C and vitamin E act synergistically to reduce free radicals and also regenerates the reduced form of vitamin E. However, the intake of high doses of vitamin C (2000 mg or more/day) has been the subject of debate for its eventual pro-oxidant or carcinogen property (Hatem et al., 2018; Labrozzi, 2020).

Beta-carotene

Beta-carotene is a pro-vitamin and converts into the active form i.e. vitamin A, a fat soluble member of the carotenoids. Beta-carotene is converted to retinol, which is essential for vision. It acts as antioxidant by quenching of singlet oxygen (Yadav et al., 2016).

Selenium

Selenium is a trace mineral found in vegetables, liver, meat, water, sea food, soil, and yeast. It helps to form the active site of several antioxidant enzymes like glutathione peroxidase (Finner, 2013). At low dose, it acts as an antioxidant, anti-carcinogenic and immunomodulator. But at higher doses it produces selenosis which is a selenium poisoning characterized by gastrointestinal disorders, hair and nail loss, cirrhosis, pulmonary edema and death (Heyland et al., 2005).

Flavonoids

Flavonoids are polyphenolic compounds present in most of the plants. Based on the chemical structure, more than 4000 flavonoids have been acknowledged and categorized into flavanols, flavanones, flavones, isoflavones, catechins, anthocyanins, proanthocyanidins. Because of their potent antioxidant activity, they are reported to prevent or delay a number of chronic and degenerative disorder such as cardiovascular diseases, cancer, memory loss, arthritis, cataract, aging, stroke, inflammation, Alzheimer’s disease, infection. Every plant contains a distinctive amalgamation of flavonoids and every plant shows very different effects on the body. The natural sources of flavonoids include green tea, grapes, apple, cocoa, ginkgo biloba, soybean, curcuma, berries, onion, broccoli, etc. For example, green tea is a rich source of flavonoids, especially flavanols (catechins) and quercetin. Catechin levels are 4-6 times greater in green tea than in black tea. Many health benefits of green tea reside in its antioxidant, anticarcinogenic, antihypercholesterolemic, antibacterial (dental caries), anti-inflammatory activities (Sathishkumar et al., 2016; Kaushik et al., 2011).

6. GENERATION OF ROS DURING DRUG METABOLISM

Cytochrome-P450 is involved in Reactive Oxygen Species (ROS) generation during phase-I of drug metabolism (Deavall et al., 2012). Substrate binding to CYP-450 occurs via the combination of one molecule of oxygen to the enzyme leading to the formation of an oxy complex (Thiboutot et al., 2003). The oxy complex thus formed is again reduced to peroxy complex that accepts two protons and produce water through intermediate reactions. Since ROS is generated during the intermediate stages of CYP-mediated biotransformation of drugs, their continuous production results in NADPH consumption by the CYP molecules (Abdel et al., 2011; Ogun, 2015). Although it was mentioned earlier that ROS is generated during the reaction of CYP-450 with its substrate, the electron-transfer chain of microsome continues to oxidize NADPH and produce ROS even in the absence of any substrate (Maxson and Mitchell, 2016). This excessive ROS generation leads to repression of CYP gene expression (Fig. 3) (specifically CYP-1A1 gene expression) is through the inactivation of the transcription factor, nuclear factor-1. Enzymes like ipoxygenase, cyclooxygenase and xanthine oxidase can also contribute to ROS production (Held, 2015; Snezhkina et al., 2020).

ROS generated during biotransformation of drugs consists of hydrogen peroxide, hydroxyl and superoxide radical. The hydroxyl radical is very reactive and can modify nitrogenous bases of DNA leading to DNA strand breakage. These ROS, particularly superoxide radical, gets protonated to form perhydroxyl radical. This radical plays a significant role in lipid peroxidation and membrane destabilization (Abdel et al., 2011). These reactive species also reacts with nitric oxide (NO) forming peroxynitrite ion (ONOO) and exerting deleterious effect on DNA, protein and lipid molecule (Koca et al., 2005; Wolf et al., 2003). Peroxisome is also a source of hydrogen peroxide but catalase present in this organelle decomposes it into water and oxygen creating a fine balance at physiological condition.

Fig. 3: Flow chart depicting CYP mediated ROS generation and transcriptional repression of CYP

7. GENERATION OF OXIDATIVE STRESS BY DRUG METABOLITES

Most of the drugs and xenobiotics, to which human beings are exposed, generate quinone metabolites. Quinones are involved in electron transport. Quinone-quinol cycle leads to oxidative stress leading to devastating effect. Many drugs are converted to quinone metabolites during biotransformation (Deavall et al., 2012). These quinone metabolites are reactive molecular species which forms adducts with macromolecules, anti-oxidant molecules like GSH and deplete the pool of antioxidant molecule like GSH thereby generating more ROS (Fig. 4). Excessive ROS generation and sequestration of endogenous antioxidant species lead to oxidative stress. Prolonged persistence of cellular stress causes sustained activation of stress responsive MAPKs ultimately manifesting into cell death (Schiffer et al., 2018).

Fig. 4: Flow chart depicting mechanism of drug metabolism induced cellular stress

Table 3- Oxidative stress producing drug metabolites

Hair follicle comprises of the epithelium of keratinocytes and a group of specified mesenchymal cells called dermal papilla. Developmental morphogenesis and postnatal growth of hair follicles are build-up with characteristic structure of epithelial-mesenchymal interaction (EMI) of these two groups of cells (Huang et al., 2017). Elevated levels of prostaglandin D2 (PGD2) have been shown to be present in the bald scalp of androgenic alopecia (AGA) patients and to functionally inhibit hair growth.

8. TREATMENT

Oxidative stress was supposed to be one of the important causative agents of androgenic alopecia, which is characterized by excessive scalp hair thinning. Hence, it can be suggested that the utilization of antioxidant molecules with some functional excipients can stop the progression of this disease (Hatem et al., 2018). Hair loss rarely needs to be treated. Many people seek treatment for cosmetic reasons, but there is no cure for alopecia and no universally proven therapy to induce hair growth. Although there are some drugs useful in alopecia and these include finasteride, minoxidil, and corticosteroids.

Finasteride

Finasteride and dutasteride are the drugs available in the market as tablet. A synthetic type II-5α reductase inhibitor can reduce the conversion of testosterone to DHT as demonstrated in Fig. 5. If it is taken for over 6 months to 1 year, at a dose of 1 mg daily, it can improve the hair count and thickness, with enhanced response. The efficacy of dutasteride 2.5 mg/day was superior to that of finasteride 5 mg/day (McElwee and Shapiro, 2012; Kaufman et al., 1998). Finasteride is reported to exhibit some side effects related to sexual functions. On continuous oral administration for long term, there can be loss of hairs, which was gained within 12 months. It is also reported to be less effective on large bald spots (Guo et al., 2017; Yu et al., 2006).

Fig. 5: Mechanism of action of finasteride

Minoxidil

Minoxidil was originally employed as antihypertensive therapy and improved the blood circulation in particular area of hair follicles and has been consequently used as a topical treatment for hair loss and is available as 2% and 5% solutions. Minoxidil use is associated with angiogenesis, vasodilation, and enhanced cell proliferation, probably mediated via potassium channel opening. Minoxidil produces some side effects when it comes in contact to skin with dermatitis and a transitory shedding with starting first 4 months of use. It helps to increase blood flow to hair follicles by dilating the blood vessels via increasing K+ channel (Fig. 6). Hence, it provides more nutrients to the hair follicle and promotes growth (Kumar et al., 2018; Sheikh et al., 2015; Bilandi et al., 2017).

Fig. 6: Mechanism action of minoxidil

Corticosteroids

Corticosteroids act by suppressing immune system. This is useful in androgenic alopecia because the condition is thought to be caused by the immune system damaging the hair follicles.

Other medications

Caffeine is used in the treatment of hair loss and stimulation of hair growth. It also inhibits phosphodiesterase (PDE) that is responsible for degradation of cAMP. This increases intracellular levels of cAMP and stimulates cellular metabolism. Furthermore, caffeine causes vasodilation and, thereby, increases blood supply to the follicles (Ramezani et al., 2018). Cyclosporin's main effect is to lower the activity of T-cells; it does so by calcineurin–phosphatase pathway and preventing the mitochondrial permeability transition pore from opening (López-Ongil et al., 1998). Cyclosporin binds to the cytosolic protein cyclophilin (immunophilin) of lymphocytes, especially of T cells. This cyclosporin—cyclophilin complex inhibits calcineurin, normally responsible for activation of transcription of interleukin-2. In T-cells, activation of the T-cell receptor normally increases intracellular calcium, which acts via calmodulin to activate calcineurin. Calcineurin then dephosphorylates the transcription factor NF-AT (nuclear factor of activated T-cells), which moves to the T-cell nucleus and increases the transcription of genes for IL-2 and related cytokines. Cyclosporin, by preventing the dephosphorylation of NF-AT, leads to reduced effector T-cell function; it does not affect cytostatic activity (Chen et al., 2002; López-Ongil et al., 1998; Pérez de Hornedo et al., 2007). In case of sulfasalazine, sulfapyridine splits off in colon by bacterial action and active compound (5-ASA is active in ulcerative colitis) absorbed systemically and generation of superoxide radicals and cytokine liberation is suppressed (Kim et al., 2009; Couto et al., 2010). Methotrexate is a folate antagonist and is a potent immunosuppressant which markedly depresses cytokine production and cellular immunity, and has anti-inflammatory property. Azathioprene is a prodrug and is converted to 6-mercaptopurine (Elelaimy et al., 2012), that inhibit de novo purine synthesis and causes DNA damage that inhibit proliferation of T cell (Bhandaru et al., 2012).

9. CONCLUSION

The issues related to the hair including reduction in hair production (alopecia) and decreases in hair coloration (greying) have been focal in human culture since prehistoric times. Hair has been associated with youth, beauty and health, and conditions leading to hair loss prove to be a cause of immense psychological distress. Intake of food supplements and nutraceuticals including vitamins, minerals, omega fatty acids and antioxidants have been reported to play a vital role in appropriate hair follicle production as well as in immune cell function. Oxidative stress is one of the important components that plays a function in the ageing as well diminution of the hair follicle. ROS are formed by a large array of endogenous and environmental sources. The body endogenously generates defensive mechanisms, such as antioxidative enzymes and non-enzymatic antioxidative chemicals, which protect against free radicals by lowering their concentration and neutralizing them. On one side the production of free radicals escalates with age and on the other side the endogenous defense mechanisms are reduced. This imbalance causes a steady degradation of cellular and molecular structures, most definitely culminating in the ageing phenotype. The therapeutic utility of antioxidants in the treatment of androgenic alopecia has been proven scientifically and expanded in the recent years. This review presents the new approaches for prevention of oxidative stress-based alopecia.

Acknowledgement

The authors are thankful to the University Institute of Pharmacy, Pandit Ravishankar Shukla University, Raipur for the infrastructural facilities. The authors extend special thanks to the Librarian, Pt. Sundarlal Sharma Library of the University for e-resources available through UGC-INFLIBNET. This work was supported by Pt. Ravishankar Shukla University Research Scholarship [797/Fin./Sch./ 2021].

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