Synchrotron x‐ray fluorescence analysis reveals diagenetic alteration of fossil melanosome trace metal chemistry

A key feature of the pigment melanin is its high binding affinity for trace metal ions. In modern vertebrates trace metals associated with melanosomes, melanin‐rich organelles, can show tissue‐specific and taxon‐specific distribution patterns. Such signals preserve in fossil melanosomes, informing on the anatomy and phylogenetic affinities of fossil vertebrates. Fossil and modern melanosomes, however, often differ in trace metal chemistry; in particular, melanosomes from fossil vertebrate eyes are depleted in Zn and enriched in Cu relative to their extant counterparts. Whether these chemical differences are biological or taphonomic in origin is unknown, limiting our ability to use melanosome trace metal chemistry to test palaeobiological hypotheses. Here, we use maturation experiments on eye melanosomes from extant vertebrates and synchrotron rapid scan‐x‐ray fluorescence analysis to show that thermal maturation can dramatically alter melanosome trace element chemistry. In particular, maturation of melanosomes in Cu‐rich solutions results in significant depletion of Zn, probably due to low pH and competition effects with Cu. These results confirm fossil melanosome chemistry is susceptible to alteration due to variations in local chemical conditions during diagenesis. Maturation experiments can provide essential data on melanosome chemical taphonomy required for accurate interpretations of preserved chemical signatures in fossils.     

Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.     

Quantitative Analysis of Eumelanin and Pheomelanin in Humans,Mice, and Other Animals: a Comparative Review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole‐2,3,5‐tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

An easy‐to‐run method for routine analysis of eumelanin and pheomelanin in pigmented tissues

A procedure for analysis of melanin‐pigmented tissues based on alkaline hydrogen peroxide degradation coupled with high‐performance liquid chromatography (HPLC) ultraviolet determination of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) for eumelanin and 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA) and 1,3‐thiazole‐2,4,5‐tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic methodology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end‐capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra‐assay precision of the analytical runs was satisfactory with CV values ≤4.0% (n = 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A₂₈₀/A₂₅₄: PTCA = 1.1, BTCA = 0.6) proved a valuable parameter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies.     

Eumelanin levels in rufous feathers explain plasma testosterone levels and survival in swallows

Pigment‐based plumage coloration and its physiological properties have attracted many researchers to explain the evolution of such ornamental traits. These studies, however, assume the functional importance of the predominant pigment while ignoring that of other minor pigments, and few studies have focused on the composition of these pigments. Using the pheomelanin‐based plumage in two swallow species, we studied the allocation of two pigments (the predominant pigment, pheomelanin, and the minor pigment, eumelanin) in relation to physiological properties and viability in populations under a natural and sexual selection. This is indispensable for studying the evolution of pheomelanin‐based plumage coloration. Pheomelanin and eumelanin share the same pathway only during their initial stages of development, which can be a key to unravel the functional importance of pigment allocation and thus of plumage coloration. Using the barn swallow, Hirundo rustica, a migratory species, we found that plasma testosterone levels increased with increasing the proportion of eumelanin pigments compared with pheomelanin pigments, but not with the amount of pheomelanin pigments, during the mating period. In the Pacific swallow Hirundo tahitica, a nonmigratory congener, we found that, during severe winter weathers, survivors had a proportionally smaller amount of eumelanin pigments compared with pheomelanin pigments than that in nonsurvivors, but no detectable difference was found in the pheomelanin pigmentation itself. These results indicated that a minor pigment, eumelanin, matters at least in some physiological measures and viability. Because the major pigment, pheomelanin, has its own physiological properties, a combination of major and minor pigments provides multiple information to the signal receivers, potentially enhancing the signaling function of pheomelanic coloration and its diversification across habitats.     

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Quantitative analysis of eumelanin and pheomelanin in humans,mice, and other animals: a comparative review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole-2,3,5-tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age‐related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) to pyrrole‐2,3,5‐tricarboxylic acid (PTCA) is a marker for the heat‐induced cross‐linking of eumelanin. In this study, we examined UVA‐induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo‐oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life‐long exposure to blue light.     

A Chemist's View of Melanogenesis

The significance of our understanding of the chemistry of melanin and melanogenesis is reviewed. Melanogenesis begins with the production of dopaquinone, a highly reactive o‐quinone. Pulse radiolysis is a powerful tool to study the fates of such highly reactive melanin precursors. Based on pulse radiolysis data reported by Land et al. (J Photochem Photobiol B: Biol 2001;64:123) and our biochemical studies, a pathway for mixed melanogenesis is proposed. Melanogenesis proceeds in three distinctive steps. The initial step is the production of cysteinyldopas by the rapid addition of cysteine to dopaquinone, which continues as long as cysteine is present (1 μM). The second step is the oxidation of cysteinyldopas to give pheomelanin, which continues as long as cysteinyldopas are present (10 μM). The last step is the production of eumelanin, which begins only after most cysteinyldopas are depleted. It thus appears that eumelanin is deposited on the preformed pheomelanin and that the ratio of eu‐ to pheomelanin is determined by the tyrosinase activity and cysteine concentration. In eumelanogenesis, dopachrome is a rather stable molecule and spontaneously decomposes to give mostly 5,6‐dihydroxyindole. Dopachrome tautomerase (Dct) catalyses the tautomerization of dopachrome to give mostly 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA). Our study confirmed that the role of Dct is to increase the ratio of DHICA in eumelanin and to increase the production of eumelanin. In addition, the cytotoxicity of o‐quinone melanin precursors was found to correlate with binding to proteins through the cysteine residues. Finally, it is still unknown how the availability of cysteine is controlled within the melanosome.     

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/Pmelanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-S-cysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/Pmelanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.     

Changes in the Proliferation and Differentiation of Neonatal Mouse Pink‐Eyed Dilution Melanocytes in the Presence of Excess Tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild‐type at the pink‐eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum‐free primary culture, with or without the addition of L‐Tyr. Incubation with added L‐Tyr inhibited the proliferation of P/P melanocytes in a concentration‐dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L‐Tyr stimulated the proliferation of p/p melanoblasts–melanocytes derived from 0.5‐day‐old mice, but inhibited their proliferation when derived from 3.5‐ or 7.5‐day‐old mice. L‐Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5‐, 3.5‐ and 7.5‐day‐old p/p mice, but L‐Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid as well as the pheomelanin marker, 4‐amino‐3‐hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid, as well as the contents of pheomelanin and its precursor, 5‐S‐cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L‐Tyr increased the content of eumelanin and pheomelanin two‐ to threefold in cultured cells and media derived from 0.5‐, 3.5‐ and 7.5‐day‐old mice. These results suggest that the proliferation of p/p melanoblasts–melanocytes is stimulated by L‐Tyr, and that the differentiation of melanocytes is induced by L‐Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

An Ultrastructural Study of Melanocytes and Melanosomes in the Skin and Hair Bulbs of Rufous Albinos

We have examined hair bulb and skin melanocytes of rufous albinos from Southern Africa to further characterize this form of albinism. In the skin melanocytes we find both eumelanosomes and pheomelanosomes at various stages of melanization and, in addition, there appeared to be many aberrant incompletely melanized melanosomes. On average, rufous melanosomes are 30% smaller than normal black skin melanosomes. In the keratinocytes, the melanosomes are packaged into distinct aggregations, whereas in normal black skin, they occur singly. We suggest that the reddish skin color of these albinos is a consequence of an increase in the pheomelanin synthesis resulting in a raised pheomelanin/eumelanin ratio and that the aggregation of melanosomes results in a skin color slightly lighter than normal. In hair bulb melanocytes, only eumelanosomes were seen and these were mostly incompletely melanized. These findings correlate with our visual observations that the hair color of Southern African albinos is very pale (light brown or ginger). Based on our observations, we speculate on the possible cause of rufous albinism.     

Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography

A method for the quantitative analysis of eumelanin and pheomelanin in tissues, e.g., hair and melanoma, is described. The method is simple and rapid because it does not require the isolation of melanins from the tissues. The rationale is that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA) which may serve as a quantitatively significant indicator of eumelanin, while hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine (AHP) as a specific indicator of pheomelanin. The degradation products, PTCA and AHP, can be readily analyzed by high-performance liquid chromatography. Chemical degradations of synthetic melanins, prepared from dopa, 5-S-cysteinyldopa, and their mixtures in various ratios, gave PTCA and AHP in yields that correlated with the dopa/5-S-cysteinyldopa ratio. The PTCA/AHP ratio as well as the contents of PTCA and AHP reflected well the type of melanogenesis in hair and melanomas. The amounts needed for each degradation were 0.5 mg of melanin, 2 mg of hair, and 5 mg of tissue samples. As many as 20 samples can be analyzed within 3 working days.     

Environmental constraints for plumage melanization in the northern goshawk Accipiter gentilis

Although it is recognized that certain environmental factors are important determinants of the expression of melanin‐based traits, their influence in wild populations of animals is poorly known. One of these factors is the availability of amino acids that serve as precursors of melanins. Here we measured eumelanin and pheomelanin content in feathers of northern goshawk Accipiter gentilis nestlings, hypothesizing that, if the availability of melanin precursors is related to food abundance and habitat quality, plumage melanization should be affected by those variables. Although the eumelanin content increased with food abundance as predicted, the levels of this variable were higher in low‐quality habitats (homogeneous coniferous forests) and in nestlings in poor condition, and the pheomelanin content and eumelanin:pheomelanin ratio were lower and higher, respectively, in subpopulations where nestlings were in poorer condition. Therefore, environmental availability of melanin precursors seems to determine plumage melanization in goshawks, but our findings may also be explained by the differential effects of environmental oxidative stress on both forms of melanin, as eumelanin and pheomelanin production are favoured under high and low levels, respectively, of oxidative stress.     

Pigment types in selected color genotypes of Asiatic sheep

The types and amounts of pigments in fibers from variously colored Tajik, Hissar, and Caracul sheep were determined by three methods: high-performance liquid chromatography, electron spin resonance spectroscopy, and light microscopic evaluation of melanosomes. In both dominant and recessive black lambs the color is due to eumelanin pigment. Brown and red phenotypes are the result of interaction of AWt and EBl, EBr, or EY alleles, and these colors are caused by mixtures of eumelanin and pheomelanin in varying ratios. The HPLC and ESR measurements detected these differences in melanin type, while direct characterization of melanosomes generally failed to distinguish between melanin type or relative ratio of melanin type.     

Visible light accelerates the ultraviolet A‐induced degradation of eumelanin and pheomelanin

Exposure to excess ultraviolet (UV) A radiation induces the degradation/modification of both eumelanin and pheomelanin that may be deleterious to pigmented tissues. Although the spectral distribution of solar energy comprises nearly half of visible light (VL), few studies have been conducted to examine the role of VL in the photodegradation of both types of melanin, either VL alone or in combination with UVA. In this study, we examined the effects of physiological doses of VL (150 to 300 J cm^?2) alone or in combination with a physiological dose of UVA (20 J cm^?2) in normal human epidermal melanocytes. The degradation/modification of melanin structures was evaluated by our chemical degradation—high performance liquid chromatography methods. The results show that VL accelerates UVA‐induced changes in the structural features of both eumelanin and pheomelanin, although VL or UVA alone induced only minor changes in melanin structure. The differential spectral method provides support for the additive effects of VL.     

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

The distribution of cadmium,copper, lead,zinc and calcium in the tissues of the earthworm Lumbricus rubellus sampled from one uncontaminated and four polluted soils

Summary The tissue distribution of Cu, Cd, Pb, Zn, and Ca in the earthworm Lumbricus rubellus living in non-polluted and heavy-metal polluted soils was investigated. Cd, Pb and Zn were primarily accumulated within the posterior alimentary canal. As the whole-worm Pb burden increased, the proportion of the metal accumulated within this tissue fraction increased. A similar pattern was found for Zn. By contrast, 70%–76% of the Cd burden was found in the posterior alimentary canal, irrespective of the whole-worm Cd content. The accumulation of Cd, Pb and Zn primarily in the posterior alimentary canal prevents dissemination of large concentrations of these metals into other earthworm tissues, and may thus represent a dextoxification strategy based on accumulative immobilisation. Cu was distributed fairly evenly in the tissue fractions investigated. There was no evidence of sequestration of this metal. The apparent lack of a detoxification strategy may contribute to the well-known susceptibility of earthworms to low environmental Cu concentrations. Indeed, earthworms from the site of highest soil Cu (Ecton) were markedly smaller than those from the other sites sampled. The highest Ca concentrations were found in the anterior alimentary canal, and were related to calciferous gland activity. A large proportion of Ca was also stored as a physiologically available pool in the posterior alimentary canal. Despite huge variations in soil Ca concentrations, the body wall Ca levels were fairly similar in L. rubellus from all the study sites. Thus, L. rubellus may become physiologically adapted to soils of exceptionally low Ca concentration. The observations are discussed in the context of the merits of analysing specific tissues, rather than whole organisms, for the purpose of monitoring metal bioaccumulation.