Experimental observations in the rat on the ... - Wiley Online Library

Int. J. Exp. Path. (2001) 82, 35±41

Experimental observations in the rat on the influence of cadmium on skin wound repair ALAN B.G. LANSDOWN 1 , B. SAMPSON 2 AND A. ROWE 3

1 Division of Investigative Sciences, Department of Chemical Pathology, Imperial College School of Medicine, London, UK, 2Trace Metals Laboratory, Department of Clinical Chemistry,

Charing Cross Hospital, London, UK and 3Department of Clinical Pharmacology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK

Received for publication 31 January 2000 Accepted for publication 7 November 2000

Summary. Wound healing in the skin depends upon the availability of appropriate trace metals as enzyme cofactors and structural components in tissue repair. The present study forms part of a series of experimental investigations to examine the influence of xenobiotic elements with no known nutritional function and which are known to compete with essential trace metals. It was designed to investigate further the importance of trace metals in wound healing as an aid to wound management and to identify mechanisms of nonhealing which constitute a major problem in human medicine. Surgically induced skin wounds in young adult male Wistar rats were exposed topically to 0.2 ml of 0.01, 0.10 or 1.0% cadmium chloride (aq.) daily for up to 10 days. Control wounds received de-ionized water only. Wounds exposed to cadmium chloride at 0.01 or 0.10% healed in a similar fashion to controls and exhibited a comparable histological profile with metallothionein distribution. Wounds receiving 1.0% cadmium chloride failed to heal or fully re-epithelialize within 7 days and animals were humanely killed. They showed a persistent mass of inflammatory cell infiltration, oedema, wound debris and aberrant epidermal cell growth. Metallothionein concentrations in the epidermis and fibroblasts of the papillary dermis increased greatly by 5 days postwounding and remained high through the observation period. Cadmium was identified in the liver, kidney and wound sites. In the wound, 1.0% cadmium chloride induced statistically significant (P . 0.001) changes in local concentrations of zinc and calcium at key stages in the healing process, and as a consequence disturbed the trace metal balance necessary for normal wound repair. Zinc levels were increased twofold after 7 days, but calcium was markedly reduced. Local changes in the distribution of metallothionein indicate interaction of cadmium and trace metal carrier proteins as a probable mechanism for impaired wound healing. The cytotoxicity of cadmium is considered to be largely responsible. Keywords: cadmium, skin wound, healing, zinc, metal ions Correspondence: Dr A. B. G. Lansdown, Skin Research and Wound Healing Laboratory, Clinical Chemistry, Division of Investigative Sciences, Imperial College School of Medicine, London, W6 8RP, UK. E-mail: [email protected] Q 2001 Blackwell Science Ltd

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A.B.G. Lansdown et al. Introduction Trace metals have a major role in tissue repair systems as cofactors in metalloenzyme systems and as structural components (Lansdown 1995). Recent studies in the rat model have demonstrated that as wounds heal, local concentrations of zinc, calcium, copper and magnesium change according to the phase in the wound healing cascade and associated biochemical events (Lansdown et al. 1999). Clinical observations in humans and experimental studies have demonstrated that deficiencies in the availability of these metals, imbalance in local concentrations, or defects in metabolism are potential causes of defective or nonhealing wounds (Moynahan 1974; Heng et al. 1993). The present study forms part of a series of experiments designed to investigate the action of xenobiotic metals with no known nutritional or trace metal value on wound healing and their interaction with trace elements with a defined role. We have examined the influence of silver, which has a long established role as an antiseptic in wound healing. This metal was shown to induce the zinc carrier protein, metallothionein, and to enhance local concentrations of zinc through the inflammatory and proliferative phases of wound healing; the repair process being accelerated (Lansdown et al. 1997). This has clear clinical implications in the development of therapeutic aids for wound healing. Lead, on the other hand, which is known to interact with zinc, magnesium and calcium, was not found to influence wound repair at cosmetic concentrations (Lansdown, 2000). We now report observations on the action of cadmium on wound healing. Cadmium is a bivalent metal with no known physiological or trace metal value in biological systems (Schroeder et al. 1967; Friberg et al. 1974; Philipp 1985). It is chemically similar to zinc and the two metals interact to a large extent in geochemical and biological systems (Elinder 1992; Waalkes & Perantoni 1988). Cadmium, like zinc, strongly induces and binds metallothionein (Waalkes & Goering 1990; Kloth et al. 1995). In earlier work, cadmium applied topically to rodent skin was found to increase local concentrations of zinc by up to 30% (Lansdown & Sampson 1996). Cadmium is a toxic metal but its cytotoxicity is mitigated through the protective action of metallothionein binding (Waalkes & Goering 1990; Liu et al. 1999). After prolonged exposure, this protective mechanism becomes saturated leading to increases in unbound cadmium and symptoms of cadmium toxicity. Alopecia with reduced DNA-synthesis, dystrophic hair papillae, depigmentation and defective hair root sheath formation 36

provide evidence for cadmium toxicity in human patients (Pierard 1979). The ability of cadmium to alter the availability and metabolism of zinc has major implications in wound healing where zinc metalloenyzymes are involved in epidermal cell replication, matrix metallo-proteinases and collagen synthesis (Pories et al. 1967; HallboÈoÈk & Lanner 1972; Lindemann & Mills 1980; Lansdown et al. 1999). On the other hand, excess zinc may inhibit the metabolism of other bivalent trace metal ions like calcium and magnesium, which are obligatory for normal wound healing, and impair wound repair (Heng et al. 1993; Presta et al. 1995). Material and methods Animals Male inbred Wistar rats (180 1 10 g body weight) were used in all experiments. They were bred under strictly controlled barrier-maintained conditions and supplied by Harlan (UK) Limited (Bicester, Oxfordshire, UK). Animal husbandry was consistent throughout with the guidelines of the Animals [Scientific Procedures] Act 1986. Thus, the rats were housed in groups of five in plastic solid-bottomed cages provided with sterile dust-free bedding and temperatures of 22 ^ 2 8C, relative humidity of 45±55%, and 12 h:12 h day/night cycles. A pelletted small rodent maintenance diet (CRM, Special Diet Service, Witham, Essex, UK) and freshly de-ionized water were available ad libitum. Cadmium concentrations of the diet and bedding as estimated by the manufacturers were , 0.01 mg/g. Zinc concentrations were in the range 55±60 mg/g. The diet was sterilized by irradiation at 2.500 Grays. Chemicals The cadmium chloride (Sigma Chemical Company Limited, Poole, Dorset, UK; minimum purity ± 99%) solutions of 1.0%, 0.1%, and 0.01% were prepared in double de-ionized water and stored at 4 8C until required. Sterile double de-ionized water served as a negative control. Experimental procedures Animals were anaesthetized by a single injection of pentabarbital sodium (Sagatalw, RhoÃne-Merieux, Harlow, Essex, UK), 35±40 mg/kg body weight and whole thickness skin wounds (15 mm long) induced surgically in the closely shaved skin of the interscapular region

Q 2001 Blackwell Science Ltd, International Journal of Experimental Pathology, 82, 35±41

Cadmium in wound healing (Lansdown & Pate 1993; Lansdown et al. 1999. Wounds were sutured at three equidistant sites using polyamide monofibre (Ethicon Limited, Edinburgh, UK). Sterility was maintained using 70% ethyl alcohol. To standardize procedures, all operations were conducted within the period 09.00±10.00 h. In initial experiments, groups of five wound-bearing rats were treated topically with 0.01 or 0.1% aqueous cadmium chloride (nonirritant); control animals received double distilled water only. Aliquots of 0.2 mL of cadmium solution were applied evenly to wound sites and the tissues allowed to dry naturally. Subsequently, wound sites were retreated with cadmium chloride solution at intervals of 24 h; animals being given light anaesthesia (30 mg/kg. pentabarbital sodium) to maximize absorption of the cadmium ion and to minimize loss of the material through animals licking wound sites. During the treatment, wound sites were examined daily for evidence of haemorrhage, ulceration, scab formation, suture loss or other changes. Cadmium-treated and control rats were killed by carbon dioxide asphyxiation after 10 days and wound sites excised. The tissue was preserved in 10% phosphate buffered formalin for histology and haematoxylin and eosin stained sections. A small sample of each wound site was preserved in cold 4% paraformaldehyde in phosphate buffered saline (2 4 8C) and metallothionein demonstrated in cryostatically prepared sections (6õÁm) exposed to monoclonal mouse antibody for 2 min as detailed in the DAKO Information Sheet (Lansdown et al. 1999). The antibody was diluted to 1 : 150 in 0.1% bovine serum albumin in phosphate buffered saline. The antibody was a mouse antihorse (E clone type) kindly donated by DAKO Laboratories (Copenhagen, Denmark) (Cat. No. M/ 0639). It is inhibited specifically by glutaraldehyde polymerized human, horse, sheep and rat metallothioneins (MTI and II) suggesting that it is directed against single and highly conserved epitopes (DAKO Information Sheet). A sample of each wound site, including tissue within 1.5 mm of the initial incision line, was cryopreserved at 2 208C for cadmium and zinc analysis. Samples of liver and kidney were also preserved for cadmium determination. In a second study, 20 wound bearing rats were treated with 1.0% cadmium chloride daily and euthanized after 2, 5, 7 or 10 days. Comparable numbers of control animals received de-ionized water only. Wound sites and samples of liver and kidney were taken as above at autopsy for cadmium analysis, histology and immunocytochemistry. To assess the influence of absorbed cadmium on key bivalent trace metals at the wound site,

tissue digests were analysed for calcium, magnesium and zinc. Metal analysis Tissue samples were digested in concentrated nitric acid and the digests diluted to an appropriate volume with double de-ionized water for trace metal determination (Lansdown & Sampson 1997). Samples were analysed for cadmium by electrothermal atomization atomic absorption spectrophotometry and for zinc by flame atomic spectrometry using a Unicam 939 instrument (ATI Cambridge, UK). Calcium and magnesium were estimated by atomic absorption spectrometry. Metal analysis was calculated on the basis of wet tissue weight. Results All animals recovered well from the surgical procedures and no fatalities were recorded. Wounds dried within 30 min and sutures remained in place for at least 24 h and were not subject to biting or other disturbance at any time. Macroscopic observations Wound healing in control rats progressed with crusting and scab formation along the suture line lasting for approximately 5 days. Thereafter, wounds re-epithelialized and showed a modest to advanced hair re-growth by 7 days. Many sutures had been exteriorized and lost by this stage although evidence of suture insertion points persisted for at least 10 days. Minimal differences were seen between control wounds and those exposed to cadmium chloride at 0.01 or 0.1%. In contrast, skin wounds treated with 1.0% cadmium chloride failed to heal normally. These wounds exhibited raised margins with erythema and oedematous changes. Scab and wound debris was more extensive and persisted for at least 7 days after surgery. Hair re-growth was poor and suture loss retarded. In view of the obvious lack of wound healing, rats exposed to 1.0% cadmium chloride were humanely terminated by carbon dioxide asphyxiation after 7 days treatment. Histopathology Differences in the histological profile between wounds treated with 0.01 or 0.1% cadmium chloride and controls were marginal through the 10 days following incision. After 10 days, wound sites were re-epithelialized but


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A.B.G. Lansdown et al.

Figure 1. Section through the wound site of a rat after 2 days application of 1.0% Cadmium chloride showing cell debris with inflammatory cell infiltration and a tongue of regenerating epidermis as early evidence of reepithelialization. 20 objective. haematoxylin and eosin (HE).

some epidermal hyperplasia was present in most sections. Dermal scar tissue by now consisted of a well defined fibrous core, with minimal chronic inflammatory cells. Skin wounds exposed to 1.0% cadmium chloride exhibited a retarded repair pattern. From 2 days, the wounds were characterized by a prominent central mass of inflammatory cells, cell debris and wound exudate (Figure 1). Foci of mineralization were occasionally present within the tissue debris. The intense infiltrate of lymphocytes, macrophages, monocytes and fibroblasts extended from the wound margin into the region of the panniculus carnosus muscle and hypodermis. Vascular dilatation and dermal oedema were prominent features of these wounds (Figure 2). Hyperplastic tongues of migrating epidermal cells were noted from 2 days but the cells exhibited a prominent vacuolation (Figure 3). Over subsequent days, epidermal cell growth

Figure 3. Down-growth of epidermal cells at the wound site of a rat treated for 7 days with 1.0% cadmium chloride. Note the marked cellular disorganization and intracellular oedema. 20 objective. HE.

failed to achieve re-epithelialization. The basal epidermis was disorganized, with the basement membrane poorly defined or absent in many places. In the dermis, the intense inflammatory infiltrate had receded slightly by 7 days postsurgery but was accompanied by necrotic changes and a lack of scar tissue formation. Follicular hyperplasia was noted in hair follicles adjacent to the wound site. Cadmium-related toxic changes were not appreciated in the liver or kidneys of animals exposed to cadmium chloride at 0.01±1.0% for up to 10 days. Immunocytochemistry In control wounds, metallothionein was identified mainly in basal cells of the epidermis at the wound margin and in fibroblasts of the papillary layer of the dermis adjacent to the wound (Figure 4). Local increases in metallothionein persisted from approximately 24 h until at least 5 days after wounding, but were normal after 7 days. In wounds treated with 0.01 o 0.1% cadmium chloride, metallothionein distribution was similar to that of control animals. However, wounds exposed to 1.0% cadmium chloride showed increased metallothionein reaction product in epidermal cells and in the papillary dermal fibroblasts of treated areas (Figure 5). Metallothionein reaction product was prominent in macrophages proximal to the wound site up until at least 7 days. Metal concentrations in wound sites

Figure 2. Vascular dilatation and oedema in the deep wound site of a rat treated with 1.0% aq cadmium chloride. 20 objective. HE.

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Cadmium concentrated in wound sites following topical treatment with cadmium chloride (Table 1). It was present also in the kidney and liver as evidence of percutaneous absorption (Very low concentrations of


Cadmium in wound healing

Figure 5. Metallothionein in a rat skin wound site treated with 1.0% cadmium chloride for 5 days. Note increased reaction product in epidermal cells and in fibroblasts of the papillary dermis. 10 Immunocytochemistry using DAKA monoclonal antibody.

Figure 4. Metallothionein reaction product in the basal cells of the epidermis and in the dermal fibroblasts of a control rat skin wound 5 days after surgery. 10 Immunocytochemistry using DAKO monoclonal antibody.

cadmium present in the liver and kidney of control animals are probable indications of an earlier uptake of cadmium from contaminants in the diet or bedding materials). Table 2 shows statistically significant differences in the concentrations of calcium, magnesium and zinc in skin wounds exposed to 1.0% cadmium chloride for up to 7 days. In the untreated wounds, zinc, magnesium and calcium levels increased markedly up until at least 5 days postwounding, but declined to normal by 7 days. In the 1.0% cadmium chloride-treated wounds, calcium

and zinc levels increased through the observation period but had not returned to the prewounding levels by 7 days. Magnesium remained low throughout. Thus, at the time the experiment was terminated, zinc and calcium concentrations at wound site were increased two and fourfold, respectively. Discussion Present studies demonstrate that cadmium is absorbed

Table 1. Cadmium concentrations in wound sites and in liver and kidney of rats treated topically with Cadmium chloride for up to 10 days Treatment group 0.01% CdCl2 0.10% CdCl2 1.00% CdCl2 1.00% CdCl2 1.00% CdCl2 CONTROL CONTROL CONTROL CONTROL

210 days 210 days 22 days 2 5 days 2 7 days ± 2 days 2 5 days 2 7 days 2 10 days

Cd in wound site

Cd in liver

Cd in kidney

10.250 41.400 0.138 3.864 4.772 0.014 0.015 0.016 0

0.050 1 0.022 ng/g 0.574 1 0.349 ngg n/a n/a 12.720 1 6.056 mg/g n/a n/a 0.017 1 0.015 ng/g n/a

0.125 0.842 n/a n/a 7.725 n/a n/a 0.030 n/a

1 1 1 1 1 1 1 1

1.089 11.49 0.064 0.940 0.856 0.005 0.003 0.005

ng/g ng/g mg/g mg/g mg/g ng/g ng/g ng/g

1 0.033 ng/g 1 0.308 ng/g 1 4.163 mg/g 1 0.018 ng/g

N/a ± not available

Table 2. Changes in trace metal content of skin wounds treated with 1.0% cadmium chloride for up to 7 days

Period of treatment

Calcium (mg/g)

Magnesium (mg/g)

2 days

0.288 (0.580 0.310 (0.700 0.473 (0.100

0.197 (0.290 0.197 (0.330 0.177 (0.210

5 days 7 days

^ ^ ^ ^ ^ 1

0.043 0.180) 0.042 0.220) 0.065 0.010)

1 1 1 1 1 1

0.010 0.080) 0.020 0.060) 0.020 0.020)

Zinc (õÁg/g) 26.704 (32.820 23.957 (30.930 28.467 (15.140

1 1 1 1 1 1

1.800 8.3203.720 1.580) 3.120 5.320)

() Control values Q 2001 Blackwell Science Ltd, International Journal of Experimental Pathology, 82, 35±41

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A.B.G. Lansdown et al. percutaneously from topically applied solutions of cadmium chloride at experimental wound sites and that at concentrations of 1.0% (i.e. 6,000ppm Cd) it is toxic and severely impairs wound repair. In this subacute study, cadmium chloride was found to be nontoxic at 0.1% even though some cadmium accumulated in the liver and kidney. Conceivably, this deposition may be a cause of structural and functional damage after longer exposure. Present studies indicate that cadmium absorbed into the skin influences local zinc concentrations, probably through its ability to induce metallothioneins (Margoshes & Vallee 1957). These proteins are known to have profound implications in the homeostasis and metabolism of zinc and copper, as well as in acting as cytoprotectants against toxic metals, free radicals and inflammatory changes (Hamer 1986; Matsubara et al. 1987; Naganuma et al. 1988; Bremner & Beattie 1990). Studies in metallothionein-null mice have shown a tenfold increase in toxic changes following exposure to cadmium chloride (Liu et al. 1999). In the present work, cadmium is implicated in the induction of metallothioneins at wound sites leading to a marginal reduction in zinc during the period of inflammatory/granulation tissue formation and epidermal cell proliferation (0±5 days) (Lansdown et al. 1999). However, this was followed by a marked rise in zinc concentration by 7 days, high local calcium, and massive inflammatory changes consistent with high cadmium concentrations and a nonhealing wound. The cellular implications of these raised metal ion concentrations await further investigation. Whereas at lower cadmium concentrations, metallothioneins were obviously sufficient to afford a cytoprotective effect for the tissues, this mechanism was saturated by 1% cadmium chloride resulting in profound cytotoxicity manifested by a prolonged inflammatory reaction (Lansdown & Sampson 1996). Our studies do not indicate to what extent zinc ion was available for metalloenzyme synthesis and wound healing, or was bound in metallothionein complex. Whereas topical zinc has an acknowledged therapeutic role in wound healing (Lansdown 1993, 1996), we are aware that excessive zinc and calcium in wound sites may retard healing (Lansdown & Sampson, unpublished). Recent studies have demonstrated that concentrations of essential trace metals change in skin wounds to reflect their requirements in metalloenzyme complexes in sequential events in the wound healing cascade (Lansdown et al. 1999). Since bivalent trace metals compete for binding sites on carrier proteins and in metabolic events, the balance of trace metals in the wound site is critical for the stage in healing. Imbalances 40

in the relative concentrations of calcium:zinc, zinc:copper, etc., or the presence of a xenobiotic ion like cadmium, are potential causes of impaired wound healing (Klevay 1975; Heng et al. 1993; VicÏanovaÂ et al. 1998). In the present work cadmium clearly disturbs the metabolism of zinc and calcium in the skin and alters the balance of trace metal ions at a critical stage in wound repair. The immunotoxicity of cadmium through environmental and dietary exposure is recognized in humans and experimental animals (Descotes 1992; Stelzer & Pazdernik 1983; Bernier et al. 1995). Experimental studies in rodents have demonstrated that although cadmium may lead to increased humoral responses at low concentrations, at high levels it induces a marked reduction in DNA and RNA synthesis in B-lymphocytes (Daum et al. 1993) with depressed cell mediated immunity and antibody production (Descotes 1992). In wound healing, these changes may be expected to evoke a reduction in phagocytosis and antibody response to pathogens and in part contribute to nonhealing or delayed repair through persistent infection and prolonged inflammatory reactions at wound sites (Istoris et al. 1999). In the present work, experimental skin wounds were exposed to appreciably higher concentrations of cadmium than might be encountered in most human industrial or domestic environments [the concentrations reported in the soil in the Shipham area of Somerset following the 1978 emergency were in the range 11± 998ppm (Philipp 1985)]. According to the World Health Organization (1980), people exposed to cadmium in their workplace can tolerate up to 10 mg/m3 in the atmosphere for a lifetime without showing ill-effects. Nevertheless, our results do point to the existence of a potential clinical hazard associated with cadmium exposure in humans. Although sensitivities to cadmium may differ in human and rat wounds, repair mechanisms and cytochemical events are likely to be similar (Lansdown et al. 1999; Gottrup et al. 2000). Although nonhealing wounds have not been identified in workers following occupational cadmium exposure so far, it is conceivable that a correlation has not been sought or identified. Epidemiological studies are required. References BERNIER J., BROUSSEAU P., KRYSTYNIAC K., TRYPHONAS H. & FOURNIER M. (1995) Immunotoxicology of heavy metals in relation to the Great Lakes. Environ. Health. Perspect. 103 (suppl. 9), 23±34. BREMNER I. & BEATTIE J.H. (1990) Metallothionein and trace minerals. Ann. Rev. Nutr. 10, 63±83.


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