The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.

Amphibian liver pigment cells, also known as Kupff er cells, contain various amounts of melanin, haemosiderin and lipofuscin. We used diff erent histochemical and ultrastructural methods to analyse and compare the level of hepatic pigmentation and the structure of hepatic pigment cells in the livers of three representatives of the family Proteidae; two subspecies of the hypogean Proteus anguinus (depigmented Proteus a. anguinus and pigmented Proteus a. parkelj ) and the epygean Necturus maculosus . Our analysis revealed differences at histological and ultrastructural level. While the percentage of the pigmented area and ultrastructural characteristics are similar in both subspecies of P. anguinus , great diff erences occur in the amount and structure of the pigment cells between P. anguinus and N. maculosus . Pigment cells are more numerous and compose larger pigmented clusters in P. anguinus . Th ey are structurally more heterogeneous and contain a larger amount of haemosiderin when compared to N. maculosus . Our results confi rm a high degree of variation in hepatic pigmentation among diff erent amphibian species. Because many factors infl uence the level of hepatic pigmentation in poikilotherms, diff erences among species from diff erent habitats and also among individuals of the same species are expected but are not easily explained. We propose two possible explanations for the large amount of iron present in Proteus anguinus : (1) accumulation of pigments due to the very low metabolic rate and extended lifetime; (2) large iron storage capacity as an adaptation to a low and discontinuous food supply in caves.

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.