Melanin affinity of toxic and carcinogenic substances

Bengt S. Larsson and Nils G. Lindquist
Dept of Toxicology, Biomedical Center, Uppsala University
Box 594, S-75124 Uppsala, Sweden

Using whole-body autoradiography and in vitro binding studies, we have investigated the melanin affinity of a large number of compounds (1-5). It is now well established that melanin has the property to accumulate several compounds such as various amines and probably also a number of metals, and to retain these agents, often for a very long time, even more than a year after a single administration (6). The physiological significance of melanin binding is however still poorly understood. One possibility is that melanin may protect the cells that harbour the pigment by keeping potentially harmful substances bound and slowly releasing the agents in low, nontoxic concentrations. The presence of melanin in some very sensitive tissues favours such a hypothesis. In the eye, and in the inner ear, melanin is located close to the receptor cells, in the retinal pigment epithelium and in the stria vascularis of the cochlea. These pigmented epithelial cells are involved in the nourishment of the receptor cells. In the brain, melanin is present in neurons in the extrapyramidal system, mainly in the substantia nigra and the locus coeruleus. However, this possible protection mechanism may under certain cicumstances be a threat to the cell. Thus may a long term exposure to a toxic compound with melanin affinity ultimately cause lesions in the cells. This mechanism seems to be an important factor in the development of some drug-induced ocular and inner ear lesions, and drug- and manganese-induced parkinsonism (1,4,7). Examples of compounds with melanin affinity causing such lesions are phenothiazine derivatives (ocular lesions, parkinsonism), chloroquine (ocular and inner ear lesions)(l,8) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; parkinsonism) (4,5,9). A conspicuous feature in the pathogenesis of these lesions is the degeneration of the melanin-containing cells.
Pigmented cells often seem to be subject to early ageing (1). The graying of hair,
i.e. degeneration in the melanocytes of the hair bulbs, is perhaps the most obvious example. Senile degeneration of melanin structures also occurs in the eye, the inner ear, and the substantia nigra. Such degeneration may be of importance in the etiology of certain forms of senile lesions, such as some types of cataract formation and retinal degeneration secondary to degeneration in the pigment epithelium, hearing loss secondary to strial atrophy, and idiopathic parkinsonism caused by destruction of the pigmented neurons in the substantia nigra and the locus coeruleus. These pre-senile conditions in the eye, the inner ear and the brain stem may be connected with accumulation and retention of noxious substances in the melanin of cells with a critical function in these organs. The melanin in these cells is very stable with a slow, if any, turnover. Therefore, it is possible that accumulation and retention of toxic agents may go on for years or even decades. Previously, the attention to melanin affinity was mainly focused on drugs. But some studies have also been dealing with the binding of metal ions to melanin, largely in vitro (10-13), but also in experimental animals (7111,14). The knowledge in this regard, however, is so far incomplete, and since may metals are potent poisons, we have started a series of experiments to investigate the uptake of various metal ions in pigmented tissues after chronic exposure.
It has been found that melanin affinity of certain compounds is very high compared with others, and the substances showing the highest affinity are mainly organic amines and metal ions (2,13,15). Melanin is a polyanion. rich in negatively charged groups such as carboxyl groups and semiquinones (16-18). Substances with cationic properties (e.g. amines and metals) are bound to the melanin by ionic interaction (15,19). Both aliphatic (20) and aromatic amines are bound, especially the polycyclic amines. In the latter case, the ionic interaction is probably strengthened by other attractions, such as van der Waal's forces at the apposition of the aromatic rings of the compounds and the indole monomers of the melanin (15). The involvement of charge-transfer interaction has also been indicated for certain electron-donating substances, e.g. chlorpromazine (15,19), as well as hydrophobic interaction in some cases (21,22). The binding is normally complex -Scatchard analyses have shown that more than one binding class is involved for individual substances, including metals (15).
A prominent feature of the lesions caused by substances with melanin affinity is that, as mentioned above, the histologic changes are found in the melanin-containing cells, and in adjacent tissues, such as receptor cells. The effects are mainly chronic and related to high dose/long-term exposure. The entire manifestation of the lesions may occur after cessation of the offending substance, even years after discontinuance of the exposure (23). It is also possible that various substances, which are retained in the pigmented tissues, may cause additive adverse effects. The histopathologic changes in the melanin-containing cells are usually characterized by enlargement of the cells and a marked increase of the melanosome number. Later, degenerative changes occur with release of meianosomes and other cellular debris, which migrate into surrounding tissues. In the eye, e.g., pigment deposits can be seen in the retina, and atrophy of the photoreceptors (for review of the melanin-related histopathology, see ref. 1). The mechanism behind the development of the lesions is possibly a combination of selective retention (i.e. melanin affinity) and toxicity. The melanin serves as a chemical depot, from which the stored chemicals slowly become released with prolonged increase of the cytoplasmic concentrations. The degenerative course is ultimately determined by the intrinsic toxicity of the individual substance. It has also been proposed that, in some cases, the interaction per se between a substance and the melanin might change the chemical properties of the melanin with alteration, or even loss, of its physiological functions - there are some evidence for a protective role of the melanin in pigmented cells, e.g. as a sink for free radicals or excited, and potentially harmful, species (for review, see ref. 2).
The melanin affinity of certain substances may also be connected with melanoma induction. The main etiological factor behind malignant melanoma is apparently UV exposure, combined with, e.g., various phenotypic risk factors. But some  epidemiologic studies and results from animal experiments have indicated that also chemical carcinogenesis may be involved in the etiology of the disease (e.g. ref. 24). In autoradiographic distribution studies on mice it has been found that some carcinogenic substances are retained in melanin-containing tissues due to melanin affinity, e.g. aflatoxin B1 (22), tobacco-specific N-nitrosamines, benzidine, polycyclic hydrocarbons (dimethylbenz(a)antracene and benzo(a)pyrene), and some food pyrolysis products (25).
In screening studies on isolated hair melanin we have recently identified more than 30 additional carcinogenic compounds with melanin affinity in vitro (Larsson & Roberto, unpublished results). The results so far obtained should exhort a methodical examination of the possible connection between melanin affinity and malignant melanoma, both epidemiologically and experimentally. A problem in this regard is that most studies on chemical carcinogenesis have routinely been performed in albino animals, which are refractory to melanin-related risks - therefore the use of pigmented animals is necessary.

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