Differential effects of dopamine melanin on norharman-induced toxicity in PC12 cells.

The food contaminant norharman structurally resembles MPTP a compound that selectively damages pigmented brain areas. Both compounds are sequestered and retained in melanin-containing neurons. The aim of the study was to examine whether intracellular melanin can modulate the toxicity of norharman in melanin-loaded PC12 cells. Dopamine melanin protected against norharman-induced upregulation of grp78, activation of caspase 3 and necrosis at low concentrations (5 and 50 microM). In contrast, at a high conentration (500 microM) there was a significantly increased expression of grp78, hsp90 and caspase 3 and a disassociation of melanin aggregates leading to dispersal of granules to swollen neurite terminals. In human populations, a long-term low-level exposure to toxicants with a high affinity to melanin will probably result in accumulation in melanin-containing neurons in vivo. Our data suggest that accumulation of a neurotoxicant in melanin-loaded cells may lead to increased cell stress, apoptotic signaling and disassociation of melanin aggregates.

Long-term retention of neurotoxic beta-carbolines in brain neuromelanin.

beta-Carbolines show structural resemblance to the neurotoxic N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and are metabolized to mitochondrial toxicants. Humans are continuously exposed to low levels of beta-carbolines through cooked food, coffee, alcoholic beverages and tobacco smoke. beta-Carbolines have previously been detected in higher levels in the pigmented substantia nigra than in the cortex of humans. The distribution of 3H-labelled harman and norharman in the brain of pigmented and albino mice and in frogs (a species having neuromelanin) was studied by tape-section and light-microscopic autoradiography. Furthermore, the binding of these beta-carbolines to dopamine-melanin and melanin granules from Sepia officinalis was examined. The results revealed a high affinity binding to melanin and a long-term retention (up to 30 days) in pigmented tissues, including neuromelanin-containing neurons of frogs after a single injection. The role of long-term exposure to food-related beta-carbolines and a retention of these compounds in pigment-containing neurons in the induction of idiopathic Parkinson's disease should be further considered.

Increased pigmentation of iridial melanocytes in primates induced by a prostaglandin analogue.

The melanocytes in the mammalian eye have been thought to produce melanin only during fetal development and in the very young individual. The recent discovery that latanoprost, a prostaglandin analogue used in the treatment of glaucoma, causes increased pigmentation of the iris in monkeys and in humans indicates that the iridial melanocytes can produce melanin in adult individuals. Using microautoradiography of HG-(3)H-methimazole, a false melanin precursor, we observed in an earlier study that there seems to be an ongoing melanogenesis in adult mice in the iridial melanocytes and in the iridial pigment epithelium. In the present study latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinor-PGF(2alpha)-isoprop yl ester) was applied once daily to the right eye of seven cynomolgus monkeys; the left eye served as an untreated control. Two animals developed clear-cut increased pigmentation of the iris in the treated eye during the first three months of treatment. These animals were injected intravenously with G-(3)H-methimazole and were killed 24 hr after the injection. The eyes were removed, fixed in 4% formalin supplemented with 10% acetic acid and embedded in paraffin or Polybed 812. Sections from the eyes were used for microautoradiography and light microscopic examination. A high uptake of radioactivity was observed in a few melanocytes in the iris of the untreated eye. There were also a low uptake in the melanocytes in the stroma of the ciliary body and the choroid. No accumulation was observed in the iridial or retinal pigment epithelium. In the iris of the treated eye the only observed difference from the untreated eye was an increased amount of melanin in the iridial melanocytes and an increased uptake of radioactivity in a great number of these cells. Thus it seems likely that treatment with latanoprost in some individuals causes an increase of the low normal melanin synthesis in iridal melanocytes.

Age-related melanogenesis in the eye of mice, studied by microautoradiography of 3H-methimazole, a specific marker of melanin synthesis.

Whether melanogenesis occurs in adult eyes is still a matter of controversy. It has been widely held that the pigment epithelial cells are fully melanized at birth, and that the uveal melanocytes cease their melanin production in the very young individual. Therefore there should be no turnover of melanin in the adult eye. A number of studies have, however, demonstrated that the enzyme involved in melanin synthesis, tyrosinase, seems to be active also in the adult eye. The recent observation that a prostaglandin analogue, used in glaucoma therapy, caused increased iridal pigmentation in the treated eye, but not in the untreated eye, of adult monkeys and in humans, indicate that the adult eye at least has the capacity to produce melanin. In the present study 3H-methimazole, a false melanin precursor, was administered to a series of DBA-mice, 3 weeks to one year of age. The eyes were removed 24 hr after a single i.p. injection of 3H-methimazole. Using microautoradiography the incorporation of radioactivity was studied in X-ray film covered sections comprising the entire eye. A very selective accumulation of radioactivity was seen in uveal melanocytes and in the pigment epithelial cells in the iris and the ciliary body. The level in the retinal pigment epithelium was low in the eyes of all ages. No uptake was seen in any non-pigmented ocular tissue. The most pronounced accumulation was seen in the pigment epithelium and melanocytes in the iris of the young mice, but some activity was seen in these cells also in the older mice. The presence of immature melanosomes seen in electron micrographs from iridal pigment cells and melanocytes of one year old mice indicate that new melanosomes are formed in these cells also in adult animals. The results of this study thus strongly indicate that there seems to be an active melanin synthesis in the adult eye of the mouse, most pronounced in iridal melanocytes and in the iridal pigment epithelium.

Uptake of manganese and cadmium from the nasal mucosa into the central nervous system via olfactory pathways in rats.

In the olfactory epithelium the primary olfactory neurones are in contact with the environment and via the axonal projections they are also connected to the olfactory bulbs of the brain. Therefore, the primary olfactory neurones provide a pathway by which foreign materials may gain access to the brain. In the present study we used autoradiography and gamma spectrometry to show that intranasal instillation of manganese (54Mn2+) in rats results in initial uptake of the metal in the olfactory bulbs. The metal was then seen to migrate via secondary and tertiary olfactory pathways and via further connections into most parts of the brain and also to the spinal cord. Intranasal instillation of cadmium (109Cd2+) resulted in uptake of the metal in the anterior parts of the olfactory bulbs but not in other areas of the brain. This indicates that this metal is unable to pass the synapses between the primary and secondary olfactory neurones in the bulbs. Intraperitoneal administration of 54Mn2+ or 109Cd2+ showed low uptake of the metals in the olfactory bulbs, an uptake not different from the rest of the brain. Manganese is a neurotoxic metal which in man can induce an extrapyramidal motor system dysfunction associated with occupational inhalation of manganese-containing dusts or fumes. We propose that the neurotoxicity of inhaled manganese is related to an uptake of the metal into the brain via the olfactory pathways. In this way manganese can circumvent the blood-brain barrier and gain direct access to the central nervous system.

Distribution of 1-(3H)-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) in the frog: uptake in neuromelanin.

The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of pigmented monoaminergic neurons of the brain, mainly in the substantia nigra. Primates and amphibians, whose nerve cells contain melanin, have shown a higher sensitivity for the toxic effects of MPTP than species which are lacking neuromelanin, e.g. rodents. In the present study the distribution after intraperitoneal injection of 3H-MPTP in frogs (Rana temporaria) was studied by whole-body autoradiography. Histochemical staining methods for melanin were used in order to identify the pigment in various tissues. Melanin-containing nerve cells were present bilaterally in the ventral motor parts of the frog brain. Melanin was also found in the meninges, around the cerebral ventricles and the aqueducts, and in the eyes, skin and liver. The results from the autoradiographic study of 3H-MPTP revealed a high accumulation and retention in all melanin-containing structures up to 15 days after administration (the longest survival time). The pigmented tissues showed the highest concentration of radioactivity in the body at all survival times. The MPTP-induced destruction of pigmented nerve cells may be related to the binding and storage of MPTP and/or its metabolites in neuromelanin, causing toxic cytoplasmic concentrations through the continuous release of substance from the melanin depot.

Pigmentation of kidneys and lymph nodes of mesocolon in rats fed diets containing the laxative danthron.

The tumor-promoting activity of the anthraquinone laxative danthron was studied by giving 3 groups of male rats a single subcutaneous injection of the colon tumor-inducing agent 1,2-dimethylhydrazine (DMH). After 1 week, the animals were fed diets containing 0, 600 or 2400 ppm of danthron for 26 weeks. Two other groups of rats were included in the study; one received no treatment while the other was given danthron only. Altogether 9 tumors were observed among animals given DMA with or without danthron. The incidence of colon tumors was higher in animals receiving DMH and danthron than in those given DMH only (5/60 vs. 0/30), but this difference was not statistically significant. The kidneys and lymph nodes of mesocolon were enlarged and showed a yellowish-red and brown discoloration, respectively. The pigment mostly displayed a PAS-positive reaction but contained no lipid as determined by several staining procedures. The available evidence suggests that the pigment is drug-derived.

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: accumulation in neuromelanin.

The herbicide paraquat has been suggested as a causative agent for Parkinson's disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinson's disease.

Disposition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice before and after monoamine oxidase and catecholamine reuptake inhibition.

The distribution of radioactivity in pigmented mice after a single intravenous injection of 1-(3H) methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) was studied by quantitative whole body autoradiography and liquid scintillation counting. Pretreatment with the monoamine oxidase inhibitors clorgyline, pargyline and deprenyl, or the catecholamine reuptake inhibitor nomifensine was performed 30 min. prior to the 3H-MPTP administration. A high uptake of radioactivity was observed in the striatum, nucleus accumbens, midbrain area and locus coeruleus, and also in the adrenal medulla. This uptake was inhibited by deprenyl or pargyline pretreatment, but not after clorgyline or nomifensine pretreatment. An extensive uptake which was not influenced by deprenyl or pargyline treatment was found in the melanin-containing tissues of the eye. This accumulation is due to the melanin affinity of MPTP and its metabolites. A comparatively rapid elimination from the brain of MPTP and its metabolites was observed, which may be due to the lack of neuromelanin in mice.

Neuromelanin and its possible protective and destructive properties.

The function of neuromelanin is not known, but some properties of the pigment suggest a protective action. Its unique ability to accumulate and retain several compounds, such as various amines and a number of metals, may protect the pigment-containing neurons from high exposure to harmful substances. This possible mechanism of protection may however in certain instances be of a double-edged nature, as accumulation of neurotoxic agents with a high melanin affinity may cause toxic concentrations in the neuro-melanin-containing cells. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) seems to be such a compound, as it has been found to preferentially destroy neuromelanin-containing cells. The degree of MPTP neurotoxicity seems to be related to the amount of neuromelanin present in the particular species. It is possible that also manganese, which is known to cause an extrapyramidal disorder resembling Parkinson's disease, causes injury to neuromelanin-bearing neurons due to its melanin affinity. This mechanism may be involved in other forms of chemically induced Parkinsonism and possibly also in idiopathic Parkinson's disease, although the offending agent remains to be discovered.

Influence of monoamine oxidase inhibitors and a dopamine uptake blocker on the distribution of 11C-N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 11C-MPTP, in the head of the rhesus monkey.

N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP, is a neurotoxic substance known to induce a parkinsonian syndrome in primates. The distribution of intravenously injected 11C-labelled MPTP (11C-MPTP) in the head of Rhesus monkeys was studied by means of positron emission tomography, PET. The influence of pretreatment with two monoamine oxidase (MAO) inhibitors, namely pargyline and clorgyline, and a dopamine uptake blocker, nomifensine, on the distribution was also evaluated. The 11C-radioactivity was taken up in all brain regions and maximum radioactivities were found 3-8 min after intravenous administration of MPTP. The 11C-MPTP-derived radioactivity showed a constant value throughout the study period in areas corresponding to the striatum and mesencephalon in monkeys not pretreated and in monkeys pretreated with clorgyline and with nomifensine. Pargyline pretreatment, however, resulted in consecutive elimination of 11C-MPTP-derived radioactivity from the different brain regions with half-lives of 40-60 min. The total radioactivity in blood was also higher after pargyline pretreatment indicating successful inhibition of metabolism. The eyes and temporal muscle each showed radioactivity values of the same order in all monkeys irrespective of pretreatment. The results support findings by other authors that MPTP was rapidly converted in the brain to a reactive metabolite which concentration remained constant in the brain during the PET study. Pargyline in the dosage used is known to be a non-selective MAO inhibitor and it prevented the metabolism of 11C-MPTP to the products retained in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in experimental animals studied by postiron emission tomography and whole body autoradiography.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a selective potent neurotoxin which has induced a syndrome similar to parkinsonism both in man and in monkeys. At autopsy degeneration of pigmented nerve cells in the pars compacta of the substantia nigra has been confirmed. The regional distribution of intravenously administered 1-(11C-methyl)-4-phenyl-1,2,3,6-tetrahydropyridine (11C-MPTP) in the brain of Rhesus monkeys was studied by positron emission tomography and the whole body distribution in mice was documented by autoradiography and by impulse counting of selected tissues. A very rapid and high uptake of 11C-MPTP derived radioactivity was seen in areas corresponding to striatum and midbrain, including the substantia nigra area. No elimination from these regions was seen during the study period of 2 h. The uptake was in the order of 7-8 times the homogenous distribution of the radioactivity in the monkey. The uptake was generally high also in other regions of the brain, but there some elimination could be distinguished. Pretreatment of the monkey with spiperone, a selective dopamine receptor antagonist, did not alter uptake nor the kinetics of the 11C-MPTP derived radioactivity. Thus 11C-MPTP does not have a high affinity for postsynaptic dopamine receptors. A remarkably high uptake of 11C-MPTP derived radioactivity was seen in the eye of the monkey. The selective uptake of radioactivity in the eye was also confirmed in pigmented but not in albino mice. The melanin affinity of MPTP may cause high intracellular concentrations of the compound or its metabolites in the melanin containing nerve cells in substantia nigra, which may explain the serious vulnerability of these neurons to MPTP.

Ototoxicity of kanamycin in albino and pigmented guinea pigs. I. A morphologic and electrophysiologic study.

Ototoxic drugs of the aminoglycoside type have been shown to accumulate to melanin, suggesting a possible mechanism for their ototoxicity. The present study was undertaken by combining electrophysiologic and morphologic methods to investigate whether the ototoxicity of kanamycin is different in pigmented and albino guinea pigs. In pigmented animals a kanamycin dose of 200 mg per kilogram of body weight per day resulted in hearing loss together with loss of both inner and outer hair cells. The albino animals in the same dose group showed significantly less hearing loss and hair cell degeneration. With daily doses of 20 and 60 mg/kg/day, no difference in ototoxicity was found between the pigmented and albino animals. The results support the hypothesis that affinity of kanamycin to inner ear melanin might be responsible for the difference in ototoxicity between albino and pigmented guinea pigs.

Effects of repeated intravenous infusions of the plasticizer di-(2-ethylhexyl) phthalate in young male rats.

The effects of six iv infusions of an emulsion containing the plasticizer di(2-ethylhexyl) phthalate (DEHP) on the liver and testes were investigated in 40-day-old rats. Groups of five to six animals received the emulsion every other day in doses of 0, 5, 50 or 500 mg DEHP/kg body weight. Liver effects were studied by histological examination and by measuring bromosulfophthalein clearance, peroxisomal proliferation and certain enzymes in serum. Testicular effects were evaluated by light and electron microscopy. To investigate the possibility of an age-related effect on the testis, five 25-day-old rats were given six infusions of 500 mg DEHP/kg. Compared with control animals, the high-dose group showed a 36% increase in relative liver weight and a 41% increase in the number of peroxisomes. In Epon-embedded testicular material from animals given the highest dose, which is about 100 times the highest estimated human exposure, some altered Sertoli cells and some degenerated primary spermatocytes were observed. No age-related effect on the testis similar to that found following oral administration of DEHP was observed in this study.

Autoradiography of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): uptake in the monoaminergic pathways and in melanin containing tissues.

A recently discovered neurotoxic compound, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been found to cause a parkinsonian-like syndrome in man and monkey, but not in laboratory animals such as rat, mouse and guinea pig. MPTP seems to selectively destroy the melanin containing dopaminergic cells in pars compacta of substantia nigra. Lower mammalian species do not have melanin in these cells, which indicates that the presence of neuromelanin may be of importance for the development of MPTP-induced lesions. By means of whole-body autoradiography of 3H-MPTP in mice, accumulation and retention was observed in the dopaminergic pathways, in locus caeruleus and in structures in the medulla oblongata and spinal cord. A high uptake was also seen in melanin-containing tissues such as in the eyes of pigmented mice. MPTP has earlier been found to have high affinity in vitro for dopamine melanin, which is similar to the pigment in substantia nigra. The typical features of the MPTP-induced neurotoxicity with destruction of pigmented nerve cells and development of parkinsonism may be to to accumulation and retention of MPTP and its metabolites in these cells. In species with pigmented nerve cells, such as man and monkey, the accumulation may be much more pronounced because of the melanin affinity of MPTP and its metabolites.

Kinetics of di-(2-ethylhexyl) phthalate in immature and mature rats and effect on testis.

The testicular response of di-(2-ethylhexyl) phthalate (DEHP), as well as the kinetics of DEHP and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP), were studied in immature and mature rats. After 14 daily oral doses of 1.0 g DEHP/kg body weight to 25, 40 and 60-day-old rats, testicular damage was observed in the youngest age group only. DEHP was not found to any significant extent in the peripheral plasma after an oral dose of 1.0 g DEHP/kg body weight. High plasma levels of MEHP were found, with maximal plasma concentrations ranging from 48 to 152 micrograms/ml. The in vitro plasma protein binding of MEHP was extensive, approximately 98%, in all age groups and no age-related difference in the elimination half-life was observed. The amount of DEHP-derived material excreted in urine was twice as high in 25 as in 60-day-old rats. The mean area under the plasma concentration-time curve of MEHP was also significantly larger in 25 than in 40 and 60-day-old rats. These observations suggest that the extent of absorption, and hence total exposure to MEHP and its metabolites, is higher in young than in more mature rats after oral administration of DEHP. It seems probable that this finding is relevant to the age-related difference in the toxic effects on the testis.

Melanin affinity of manganese.

The melanin affinity of manganese was studied in vitro using melanin isolated from beef eyes or human hair, and synthetic dopamine melanin, which is known to be structurally similar to the melanin present in the pigmented nerve cells in the human substantia nigra. In addition, the uptake of manganese in melanin containing tissues in vitro and in vivo was studied by whole body autoradiography. Manganese was bound to beef eye, human hair and synthetic dopamine melanin, and was taken up in pigmented tissues in mice and a monkey. Long-time exposure to manganese may cause a chronic extrapyramidal disorder. It is suggested that manganese, due to its neurotoxicity, may cause lesions in pigment containing neurones in the substantia nigra secondary to its accumulation on the neuromelanin.