ANNALS OF MICROSCOPY Vol 8, April 2008 A Gross Anatomical ... · globally by both gender to...

ANNALS OF MICROSCOPY Vol 8, April 2008

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A Gross Anatomical and Histological Inspection of the Mammalian Integument Post-Treated With Aluminium Nitrate

Nasir Aziz* and Rabail Nasir Aziz

School of Health Sciences (PPSK), University Sciences of Malaysia. 16150, Kubang Kerian, Kelantan Darul Naim, Malaysia.*Corresponding email: [email protected]

ABSTRACT

Commercially viable patented antiperspirant agents are daily household products used globally by both gender to minimize excessive sweating or hyperhidrosis especially within the axillae, palms, soles and of the face. Among these preparations includes Botulinum toxin A, tap water iontophoresis and metal salts. While Aluminium nitrate and Aluminium chloride are used as the main metal salts in various titration concentrations. However, Aluminium nitrate, being a metal entity, can be haphazard toxic to the skin histology. This present study was aimed to observe under the research grade compound light microscope the histo-toxicological effects manifested post-application of 10% and 20% W/V solutions of aluminium nitrate that was topically applied onto the outermost surface of the ear integument of 36 Albino rabbits treated daily with the help of a soft brush. The animals were then divided into two groups comprising of 24 experimental and 12 controls. Initially half of the experimental and control animals were sacrificed after 15 days of application to observe immediate effects, whereas the rest of the animals were kept for another month without any further treatment and then sacrificed on day 45 to elucidate its delayed effects. The tissues were then routinely processed, semithin sectioned and post-stained using four different stains. The results revealed histological features of a thickened epidermis with hyperkeratosis, acanthosis and the present of epidermal micro-abscesses. While the dermis showed features of edema, increase in vascularity, decrease in sweat and sebaceous glands present and infiltration inflammatory cells. It was significantly noted that the histological features were far more severely manifested in the integument applied with 20% w/v solution as compared to that applied with 10% w/v solution. However these changes persisted in a moderate form following discontinuation of aluminium nitrate application.

Keywords: Aluminium nitrate, toxicity, skin and light microscopy

INTRODUCTION

The global cosmetic and skincare industry is a highly profitable market world wide that is concerned with utilisation of raw materials, product testing, sophisticated laboratory equipment and new technology testing that is said to focus within the safe limits parameters of cosmetics and toiletries usage development. As such, these cosmetic giant companies spend lavishly more on TV advertising than any other commercial enterprise. Consumers are introduced and used in various cosmetics and personal care products without full disclosure on its package labeling. The average person is misled to believe that such product ingredients have been adequately tested and are safe for public usage. Ironically, this is far from accurate (Hampton 1987). Moreover, males and females individuals, from infancy to old age utilize a huge multivariate variety of shampoos, body soaps, antiperspirants and skin creams preparations, some with no known peer-knowledge or concern of the preparation hazardous potential.

Dr. Colman is particularly concerned about the free giveaway and marketing of cosmetic products directly to breast cancer patients, who are acutely at risk of further exposure to estrogenic


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and mutagenic chemicals. He believes giving these free products in a hospital setting is contradictory to the Hippocratic oath -”first do no harm” (Colman).

Hyperhidrosis is the production of excess sweat of armpits, hands, feet or face (Draelos 2001). Hyperhidrosis is often exacerbated by emotional stress (Ruchinskas et al. 2002). Most of the treatment modalities used is effective only in mild cases. Local intradermal injections of botulinum toxin A, a neurotoxin blocking the cholinergic stimulus of eccrine sweat glands has proved to be an effective treatment (Lauchli et al. 2003).

Sweat is a dilute salt solution produced by eccrine sweat glands spontaneously or in response to heat, exercise and stressful events. Eccrine sweat is initially odorless but can start to smell if bacteria get a chance to break down the stale sweat. The eccrine glands are distributed over the entire body but are most numerous under the arms and on the palms and soles.

Apocrine sweat glands are located under the arms, around the breasts and in the groin. After puberty, they produce a thick secretion that contains pheromones, the “personal scent” that most people find unpleasant. In addition, bacteria that normally live on the skin break down apocrine sweat and this produces offensive body odor. Body odor is worse if there are more bacteria present or the level of apocrine sweat production is high. Antiperspirants also help to reduce apocrine sweat production.

Aluminum chemicals are used in a variety of cosmetics and personal care products. Aluminum chloride and Aluminium nitrate are used as antiperspirants in concentrations of 10% to 20%. These chemicals are toxic and capable of causing a variety of allergic reactions (Hampton 1987).

Aluminium nitrate is a chemical compound. It is a salt of Aluminium and nitric acid, and exists at standard temperatures and pressures as a crystalline hydrate, most commonly as aluminum nitrate nonahydrate, Al (NO3) 3·9H2O, with a molecular formula weight of 375.14. Aluminum nitrate is a strong oxidizing agent. Aluminum nitrate nonahydrate is used in the preparation of insulating papers, on transformer core laminates and in cathode-ray tube heating elements; as a tanning agent, antiperspirant, nitrating agent, corrosion inhibitor, catalyst in petroleum refining and in uranium extraction; and in the manufacture of incandescent filaments (Grams 1992 and Budavari 1996) leather, antiperspirants, corrosion inhibitors, extraction of uranium, and as a nitrating agent.

No to slight irritation (graded 0.35-0.54/8) was observed in rabbits following application of aluminum nitrate powder (form unspecified) under a moist gauze patch for 4 or 23 hours (Guillot et al. 1982). Evidence of skin irritation and occasional tissue death (ulceration) was observed in mice, rabbits and guinea pigs following application of a 10% solution for 5 consecutive days (Lansdown 1973).

One reviewer has concluded that there is a likely connection between long-term occupational exposure to aluminum and a specific effect, impaired co-ordination, but not other toxic effects on the nervous system or Alzheimer’s disease (Doll, 1993). One report indicates that aluminum can be absorbed through the skin of mice following long-term application of another water-soluble salt (aluminum chloride hexahydrate) (Anane et al. 1995). This study indicates that long-term skin contact may contribute to overall exposure and accumulation of aluminum in the body. The relevance of this finding to aluminum nitrate monohydrate is not known.

Decreased growth of newborn rats was observed following oral administration of 180, 360 or 720 mg/kg/day to mothers for 22 days during pregnancy and 21 days after birth. Toxic effects to the mothers would be expected to occur at these doses (Domingo et al. 1987). In another study, harmful effects on the fetus (decreased body weight and tail length) and teratogenic effects (skull and rib malformations) were observed in the presence of maternal toxicity in rats following administration of 180, 360 or 720 mg/kg/day to mothers during pregnancy (Paternain et al. 1988).

In a recent study, 10% and 20% W/V solutions of aluminium chloride were applied to rabbit skin for 15 days to see immediate effects and for another one month without treatment to see the delayed effects this revealed gross and histological changes in epidermis like acanthosis,


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epidermal microabcesses and hyperkeratosis while dermis showed oedema, vascularisation, decrease in hair follicles, sweat and sebaceous glands and inflammatory cells infiltration. The histological changes were more severe in the 20% W/V solution as compared to than of 10% W/V solution. After discontinuation of the treatment, the changes persisted in a milder form (Nasir A et al. 2006). It concludes that antiperspirants containing aluminium salts in higher concentration W/V, cause direct damage to integument if applied continuously for a long period.

OBJECTIVES

Antiperspirants are chemical agents that reduce perspiration or sweating. The active ingredients of roll-on and spray formulations are traditionally the metallic salts aluminium chloride and aluminium nitrates. These are formulated into preparations of varying strengths. More concentrated solutions are used to control excessive sweating (hyperhidrosis). These metallic compounds do have some toxic effects on skin resulting into thickening, hardening, hypersensitivity and hyper-pigmentation of the skin. Modern life styles of the public were kept unaware of utilizing antiperspirants containing metallic salts that are detrimental and of hazardous effects. Therefore the present study was conducted as an advancement of knowledge and an as evidence-base study of the toxicological effects of aluminium nitrate especially on the mammalian skin vis to vis in regulating complications manifested by exploitation by cosmetics producers.

MATERIALS AND METHODS

Thirty-six adult albino rabbits of both genders were selected and kept in the animal house located at the Postgraduate Medical Institute in Lahore Pakistan for the present study. They were fed on ordinary laboratory diet and regular access to tap water. The animals were divided into three main groups, A, B and C comprising of 12 animals in each. Group A received 10% aluminium nitrate, group B received 20% aluminium nitrate and group C received distilled water treatment. These groups were further subdivided into A1, A2, B1, B2, C1and C2 comprising of 6 animals in each. Group A1 and B1 were utilized to see the immediate effect with animals of C1 group as control and group A2 and B2 were used to find out delayed effects with animals of group C2 as control. About 4 cm skin of posterior surface of ear and gluteal region of each rabbit was used as test areas. These identified areas were then shaved 24 hours prior to the application of aluminium nitrate solution. Powdered form (98% purity) of aluminium nitrate (of E. Merk Company) was used. Solution was prepared in distilled water at 10% and 20% w/v concentrations daily before application. 0.5 ml solution was applied daily to the test areas. After the application, each experimental animal was kept in individual wooden cage until the applied solution dried up at ordinary room temperature and atmosphere. The procedure was repeated daily for the next fifteen days. Whereas, only distilled water was applied to the control animals. On the day 16 of treatment, 6 animals from group A1, B1 and C1 were sacrificed to see the immediate changes in their integument. The remaining animals of groups A2, B2 and C2 were kept without treatment and then sacrificed on day 46 of captivation. The macroscopic or gross changes of the skin were seen with the help of hand lens while the histological changes were observed after the tissues were chemically fixed in neutral buffered formalin solution and later processed. Five-micron thick sections were sectioned and stained either with Harris’s Haematoxylin and eosin satin, or Hartman’s method, or Periodic Acid Schiff and Haematoxylin stains.


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RESULTS

The experimental subgroup A1 animals showed following morphological changes.

Gross Changes The skin appears grayish brown in color, rough, moderately ulcerated, with fine crusts and hairs (Table-1).

Table. 1 Gross changes within the mammalian skin after 15 days treatment with aluminium nitrate as compared with the control group

Treatment Color Surface Crusting Scaling Ulcer Hairs

AluminiumNitrate 10% Greyish brown Rough Slight Nil Moderate Slight

AluminiumNitrate 20% Greyish brown Rough Moderate Nil Moderate Nil

Control Silvery white Smooth Nil Very fine Nil Moderate

Microscopic appearance

A. EPIDERMIS

It shows slight acanthosis features. The basal layer consists of single layer of cuboidal cells with vesicular nuclei. These cells are loosely placed and lie at different levels. The cells of upper layer are oval or flat with vesicular nuclei. This layer shows marked spongiosis. Hyperkeratosis is marked in stratum corneum. Small epidermal microabcesses are present at the ostia of hair follicles (Table- III, Fig3,4,5)

Table. III Epidermal changes within the skin as treated with aluminium nitrate.

After 15 days of treatment After one month of stoppage of treatment

Gross changes AluminiumNitrate 10%

AluminiumNitrate 20%



Hyperkeratosis Nil Slight Nil Slight

Acanthosis Slight Slight Slight Slight

Epidermal microabcesses Nil Nil Nil Nil

Spongiosis Nil Nil Nil Nil

B. DERMIS

It showed marked edema mainly in its lower part. Hair follicles, sebaceous glands and neuromuscular bundles are present but very few sweat glands are seen. Inflammatory cells are present mainly near dermo-epidermal junction. Vascularisation is not marked (Table-IV, Fig-4,5,6,)

The animals of experimental subgroup A2 shows following changes.


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Table. IV Dermal changes in the skin treated with aluminium nitrate

Changes After 15 days of treatment After one month ofstoppage of treatment

Control AluminiumNitrate 10%




Edema Nil Slight Moderate Rare Slight

Vascularisation Slight Slight Nil Slight Slight

Bullae and vesicles Nil Rare Nil Nil Nil

Inflammatory cells Nil Slight Moderate Rare Rare

Fibroblast Slight Slight Moderate Slight Moderate

Hair follicles Slight Slight Moderate Moderate Moderate

Sebaceous glands Slight Rare Rare Slight Slight

Sweat gland Slight Rare Rare Slight Slight

Gross changes

Skin is grayish brown, rough with fine crusting and ulceration (Table-II)

Table. II Gross changes in the mammalian skin after one month of stoppage of aluminium nitrate treatment as compared with the control group

Treatment Color Surface Crusting Scaling Ulcer Hairs

AluminiumNitrate10% Greyish brown Rough Slight Nil Slight Slight

AluminiumNitrate20% Brownish Rough Slight Nil Slight Nil

Control Silvery white Smooth Nil Very fine Nil Moderate

Microscopic changes

A. EPIDERMIS

It shows acanthosis. It is 3-4 cells thick. Basal layer contains polygonal cells closely packed to each other. Nuclei are rounded and darkly stained. Upper layer contains 1-3 layers of rounded or oval cells with pyknotic nuclei lying at different levels. Stratum corneum is very thin (Table-IV, Fig-7)

B. DERMIS

It shows slight edema. A large number of hair follicles and sebaceous glands are present. Very few inflammatory cells are also seen (Table-IV, Fig-8)

The animals of subgroup B1 shows following changes


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Gross changes

These are almost similar to subgroup A1 (Table –I)

Microscopic changes

A. EPIDERMIS

It shows marked hypertrophy. It consists of 5-7 layers of cells. Basal layer contains polyhedral cells. Most of these cells contain vesicular and rounded nuclei while the others have pyknotic nuclei. The cells of the upper layer are polygonal or oval in shape. Stratum corneum shows marked hyperkeratosis (Table- III, Fig-9)

B. DERMIS

It shows edema with inflammatory cell infiltration. Hair follicles and sebaceous glands are marked. Sweat glands are rare (Table-IV, Fig-10)

Experimental subgroup B2 shows following changes

Gross changes

These are like those of subgroup A2 (Table-II)

Microscopic changes

A. EPIDERMIS

It is thin, comprising of 2-3 layers of cells. Basal layer contains polygonal cells with vesicular nuclei. Upper layer contains 1-2 layers of flattened cells with flat vesicular nuclei lying at a distance from each other. Stratum corneum is thick but comparatively less than group B1 (Fig-11)

B. DERMIS

It shows edema, which is marked in the lower region. A large number of hair follicles and sebaceous glands are present (Table-IV, Fig-12)

DISCUSSION

Aluminium salts are widely used commercially in cosmetics and other medicinal preparations due to their antiperspirant and astringent properties. Their efficacy as antiperspirants has been well proved already. These salts irritate the mammalian skin immediately once applied however the intensity of their irritancy varies according to the concentration used in antiperspirant preparations. Aluminum nitrate exists in the anhydrous and hydrated forms, of which the nonahydrate (Al (NO3) 3.9H2O) is the most stable. Commercially, aluminum nitrate is available as the form of nonahydrate (Grams 1992 and Paternain 1998). However, clinically Aluminium nitrate is more irritating as compared to chloride, chlorhydrate and citrate.

Aluminium-based antiperspirants works by occluding the sweat ducts, thereby reducing the amount of sweat that is able to reach the skin’s outer surface. Aluminium salts are soluble


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Figure 1. Photomicrograph of well keratinised rabbit ear with prominent melanocytes cells present. (Group C1, H & E, x700)

Figure 2. Photomicrograph of rabbit ear showing epidermis and dermis with sebaceous glands and hair follicles. (Group C2, H & E, x700)

Figure 3. Photomicrograph of rabbit ear revealing the presentation of a slightly epidermal hypertrophy with micro-abscesses, vascularisation and dermal edema. (Group A1, H & E, x140)

Figure 4. Photomicrograph revealing various features of hyperkeratosis, acanthosis and spongiosis manifested here by the epidermis stratum. (Group A1, H & E, x700)

Figure 5. Photomicrograph showing acanthosis and hyperkeratosis of epidermis and marked edema in the dermis. (Group A1, H & E, x700)

Figure 6. Photomicrograph showing epidermal micro-abscess, hyperkeratosis, acanthosis, dermal edema and sebaceous glands. (Group A1, H & E, x700)


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Figure7. Semithin photomicrograph showing the various stratum / layers of the rabbit epidermis. (Group A2, H & E, x1400)

Figure 8. Photomicrograph of the experimental ear integument indicating an increase in vascularisation and dilated blood vessels in its dermis. (Group A2, HM stain, x350)

Figure 9. Photomicrograph of the ear integument section with epidermal hypertrophy. Noted the marked dermal edema and inflammatory cells. (Group B1, H & E, x700)

Figure 10. Photomicrograph of ear showing large num-ber of hair follicles and asinus of the sebaceous glands. (Group B1, PAS, x350)

Figure 11. Photomicrograph showing irregular hap-hazardly arranged epidermis with slight dermal edema. (Group B2, H & E, x350)

Figure 12. Photomicrograph of ear showing 4-6 strati-fied layering of the epidermis with hyperkeratosis fea-tures present. (Group B2, H & E, x350)


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as long as the formulation is in acidic (i.e., low pH). When they are applied to skin and come in contact with sweat, the pH rises causing the aluminium salts to precipitate out and form a plug over the sweat glands. Sweat continues to be produced by the sweat gland but it just isn’t able to reach the surface of the skin. The production of epidermal damage is due to the ability of the aluminium cations to denature epidermal keratin and the action of free mineral acid formed by hydrolysis of compound in water. The epidermal damage may be due to the effect of deposition of aluminium into the keratin. It was claimed that aluminium chloride does not cause epidermal damage because the aluminium was not available in a form, which could interact with keratin. It may cause adverse effects in only sensitive people (Lansdown 1973)

The physiological balance of the skin may affect the aluminium-protein interaction. Sebaceous lipids on the skin surface may change into a state of acidity and hydration thus contributing to the irritation of the skin once applied with the aluminium compounds. The aluminium-protein interaction may be accompanied by denaturation of the protein due to deposition of serum albumen in the solutions of aluminium salts. So the uptake of aluminium by the keratin may result into a chemical interaction between the metal ions and keratin leading to its denaturation. A recent study showed that aluminium salts treatment to the skin causes denaturation of epidermal keratin resulting into increased permeability of stratum corneum to substances applied topically. So the agent exposes the epidermal cells of deeper layers to injury, which is responsible for keratin denaturation. This cellular injury may result into histological changes like acanthosis, hyperkeratosis, epidermal microabcesses and spongiosis. In the dermis marked edema, inflammatory cell infiltration, vascularisation and changes in hair, sebaceous and sweat glands were noticed. These changes were dose dependent (Nasir 2006).

Present study clearly support and showed almost similar gross and histological changes within the skin integumentary once applied with aluminium nitrate. The observed changes within the skin morphology were far more severe when applied with 20% w/v solution of aluminium nitrate as compared to 10% w/v solution of aluminium nitrate. It shows that with the increase in concentration of the salt, the risk of damage to the epidermis and dermis increases. The gross and microscopic changes are more severe with 10% and 20% w/v solution of aluminium nitrate as compared to those of aluminium chloride. So 5-10% w/v solutions of aluminium chloride are much safer than 10% w/v solution of aluminium nitrate to be used as antiperspirant. The pathological changes in both epidermis and dermis persisted until the application of the aluminium chloride solutions are continued. As soon as the treatment is stopped, the changes reverted to the normal with almost complete reversion to normal skin (Nasir, 2006). In contrast to this, present study shows that pathological changes produced by 10% and 20% w/v solutions of aluminium nitrate persisted to slight and moderate extent even after one-month interval of stoppage of application.

CONCLUSION

Maintenance of a health life-style is crucial in daily well being The present study concludes that antiperspirants containing aluminium nitrate even in low dose concentrations w/v, may still cause well-defined histo-chemical damage to the skin and its appendages, especially after a prolong time period of continuous application. And if this time interval application is further continued permanent histo-changes are eminent.

REFERENCES

Anane, R., et al. (1995). Bioaccumulation of water-soluble aluminium chloride in the hippocampus after transdermal uptake in mice. Archives of Toxicology. Vol. 69, no. 8. p.568-571

Budavari, S, ed. (1996). The Merck index: an encyclopedia of chemicals, drugs, and biologicals. 12th ed. Merck and Co., Inc.,

Colman, J., Cosmetics and fragrance products pose high risk for breast cancer and other illness,


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www.cancer research america.org/ Doll, R. (1993). Review: Alzheimer’s disease and environmental aluminium. Age and Aging. Vol.

22, No. 2. p. 138-153.Domingo, J.L., et al. (1987). Effects of oral aluminum administration on perinatal and postnatal

development in rats. Research Communications in Chemical Pathology and Pharmacology. Vol. 57, no. 1 (July). p. 129-13.

Draelos, Z.D. (2001). Antiperspirants and the hyperhidrosis patient. Dermatol Ther 14, pp. 220–224

Grams, G.W. (1992). Aluminum compounds: aluminum halides and aluminum nitrate: aluminum nitrate. In: Kirk-Othmer encyclopedia of chemical technology. 4th ed. Vol. 2. John Wiley and Sons. p. 289

Guillot, J.P., et al. (1982). Evaluation of the cutaneous-irritation potential of 56 compounds. Food and Chemical Toxicology. Vol. 20, no. 5. p. 563-572

Hampton, A (1987). Natural Hair and Skin Care, Organic Press, Tampa, FL, Lansdown, A.B.G. (1973). Production of epidermal damage in mammalian skins by some simple

aluminium compounds. British Journal of Dermatology. Vol. 89. p. 67-76Lauchli S. and G. Burg, (2003). Treatment of hyperhidrosis with botulinum toxin A. Skin Therapy

Lett 8(7), pp. 1–4Nasir A, Farid Che G. (2006). Histo-morphological changes observed in the mammalian integument

post-treated with aluminium chloride: A light microscopic study. Annals of Microscopy Vol 6, April 66-73.

Paternain, J.L., et al. (1988). Embryotoxic and teratogenic effects of aluminum nitrate in rats upon oral administration. Teratology. Vol. 38, no. 3 (Sept) p. 253-257

Ruchinskas, R.A., R.K. Narayan, R.J. Meagher et al. (2002). The relationship of psychopathology and hyperhidrosis. Br J Dermatol 147, pp. 733–735.

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