Regulation of protein kinase C and ornithine decarboxylase in the epidermis of juvenile white suckers (Catostomus commersoni) by 12-O-tetradecanoylphorbol-13-acetate, 17 α-ethinylestradiol and testosterone

This study was designed to investigate whether potent regulators of mammalian protein kinase C (PKC) and ornithine decarboxylase (ODC) activity also regulate epidermal PKC and ODC activity in fish. Juvenile white suckers (Catostomus commersoni) were given single or multiple subdermal injections of testosterone, 17α-ethinylestradiol or 12-O-tetradeconylphorbol-13-acetate (TPA) dissolved in sunflower oil. Sequential activation of epidermal PKC and ODC was observed in single injection protocols. Maximal PKC activity occurred at 12–48 hr post-injection, with a corresponding increase in ODC activity in the 12–48 hr immediately following this event. In the multiple injection protocols, PKC activity was almost completely depressed after 1 week of injections, during which ODC activity was stimulated 2- to 5-fold, indicating possible differential activation of these two enzymes. Multiple injections of testosterone, 17α-ethinylestradiol and TPA induced histologically distinct epidermal hyperplasia in suckers, although this did not occur in single injection treatments. The mammalian isozymes of PKC are known to be dependent on Ca²⁺ and phospholipid for optimum activity. This study demonstrated that the fish isozyme of PKC is also Ca²⁺ and phospholipid dependent. Our results indicate that PKC and ODC may be good biochemical markers for neoplasia and hyperplasia in fish.     

Regression,proliferation and development of lip papillomas in wild white suckers,Catostomus commersoni,held in the laboratory

Synopsis Epizootics of skin neoplasms in teleost species have been documented within the Great Lakes region over the last decade. The white sucker,Catostomus commersoni, has been proposed as a sentinel species to monitor environmental health in these systems. The prevalence of skin neoplasia is elevated in white suckers and other fish taken from chemically polluted sites or from lake regions adjacent to heavy industry. Lip papillomas regress and proliferate spontaneously in captive wild suckers. It is important to investigate the relevance of these observations to papilloma etiology to determine whether the prevalence of this disease is a suitable biomarker for environmental health. White suckers were captured in the spring of 1992 and 1993 during annual spawning runs (mid to late April) at the Ganaraska River, which discharges into Lake Ontario at Port Hope, Ontario. Under crowded laboratory conditions, there was either proliferation of existing papillomas or development of new papillomas. However, in uncrowded conditions, existing papillomas either regressed completely or there was no development of new papillomas. Protein Kinase C (PKC), a proposed marker enzyme for hyperplasia and neoplasia, was used to determine if regressing and proliferating papillomas could be differentiated on the basis of biochemical activity. PKC activity was lower in proliferating papillomas, but not significantly different from papillomas sampled initially from Ganaraska River suckers. Regressing papillomatous tissue displayed a significantly higher level of enzyme activity than either proliferating or unchanged papillomas, but the PKC activity of regressing papillomas was not significantly different from that of normal lip epidermis.     

Androgen and estrogen stimulation of ornithine decarboxylase activity in mouse kidney

In the present work, the activity of mouse renal ornithine decarboxylase (ODC) from CBA female mice was used as a biological marker to detect (anti)androgenic activity of different groups of endocrine disruptors and steroids. Daily injections of testosterone or dihydrotestosterone (DHT) into 60 day old female mice for 4 days increased renal ODC activity in a dose-dependent manner that reached up to 100-fold (testosterone) or 250-fold (DHT) above the baseline when the highest dose, 200 microg/mouse, was used. Administration of flutamide concurrently with testosterone (75 microg/mouse) caused a potent decrease of ODC induction in a dose-dependent manner, suppressing the enzyme activity at the doses of 0.1 and 0.5 mg/mouse by about 88 and 95%, respectively. In contrast, estradiol at the doses of 0.5 and 1 mg/mouse induced a significant stimulation of renal ODC activity in a dose-dependent manner when it was given alone or in combination with testosterone. Using a sensitive increase in ODC activity in response to androgens as an end point, we did not detect an antiandrogenic effect of several antiandrogens, such as cyproterone acetate, spironolactone, p,p'DDE and vinclozolin. Also, none of these antiandrogens were able to change the basal level of renal ODC activity, with the exception of cyproterone acetate that at a dose of 0.1 mg/mouse stimulated ODC activity. The data obtained suggest that mouse renal ODC from CBA females is not strictly androgen-specific and cannot be used for estimation of antiandrogenic effects of compounds having an affinity to different types of receptors.     

Some aspects of hepatic function in feral brown trout, Salmo trutta, living in metal contaminated water

Brown trout, Salmo trutta, exposed to heavy metals (mainly Cd and Zn) for at least 2 years in the Eagle River, Colorado, were examined for liver size and activity of the growth-promoting enzyme, ornithine decarboxylase (ODC) and compared to trout living in an uncontaminated site. Liver-somatic index (LSI) was greater for trout living in the uncontaminated site with the LSI of females being significantly greater than that of males. The LSI for females at the uncontaminated site was greater than that of females at the contaminated site, but males were not different statistically. ODC activity in the livers of both males and females was lower at the contaminated site. However, males and females did not differ with respect to ODC activity. These data suggest that chronic exposure to heavy metals may have important implications for growth and reproduction and possibly survival. The activity of ODC in liver might serve as a useful biomarker when assessing chronic toxicity of metals to naturally reproducing fish populations.     

Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis

After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.     

Intersex and other reproductive disruption of fish in wastewater effluent dominated Colorado streams

Intersex white suckers (Catostomus commersoni) were collected in Boulder Creek and the South Platte River downstream of wastewater treatment plant (WWTP) effluent but not at reference sites. Eighty-three percent of the 60 white suckers collected downstream of the Boulder WWTP in spring and fall were female compared to 45% upstream (n=33). Only female (n=16) and intersex white suckers (n=4) were collected in the South Platte River downstream of the Denver WWTP in the spring. Gonad deformities and delayed follicular maturation were noted in some white suckers in Boulder Creek and the South Platte River downstream of the WWTP effluents. Asynchronous ovarian development was found in some female white suckers downstream of the WWTP effluents, but not upstream. The types and extent of sexual disruption differed in each river studied.     

Serum calcium concentrations in white sucker, Catostomus commersoni Lacepède, and bluegill, Lepomis macrochirus Rafinesque, in northern Wisconsin lakes: relation to pH and waterborne calcium

We measured serum calcium concentrations in mature white suckers and bluegills collected from clear-water Wisconsin lakes before spawning. The mean serum calcium concentration in female white suckers was greater in a circumneutral lake than in two naturally acidic lakes, whereas mean concentrations in fish from the two acidic lakes did not differ. Serum calcium concentrations in female bluegills from four acidic and four circumneutral lakes varied among lakes, but no relation was apparent between serum calcium level and either lake pH or waterborne calcium. In contrast, a strong negative correlation between mean serum calcium concentration and relative density of bluegill populations in the eight lakes was observed, suggesting that serum calcium levels in this species are affected by density-dependent factors. Mean serum calcium concentrations were substantially lower in mature male bluegills than in females, which is the norm prior to spawning. We hypothesized that serum calcium levels in female white suckers are depressed by exposure to pH 6.0 or less in calcium-poor waters. The utility of serum calcium concentrations, measured before spawning, as a measure of pH-related stress on populations of white suckers and other synchronous spawners merits further study. However, this measure is probably not useful for evaluating acid stress on fish species such as the bluegill, which have asynchronous reproductive cycles.     

Androgen-dependence of ornithine decarboxylase in the rat epididymis

Ornithine decarboxylase (ODC) activity was measured in epididymides of 45-day-old rats. Higher ODC activity was detected in the corpus and cauda than in the caput epididymidis. Bilateral castration for 7 days caused epididymal ODC to fall to undetectable values, whereas testosterone restored activity to normal values. The effect of the androgen was significantly inhibited by cyproterone acetate. The caput was more sensitive to the action of testosterone than were the corpus and caudal segments. Unilateral castration for 4 or 8 days did not affect ODC on the control or castrated side, but the activity fell in epididymides of both sides after removal of the remaining testis. These results show that epididymal ODC activity is androgen-dependent.     

The prevalence of Trypanosoma catostomi in white sucker (Catostomus commersoni) from southern Ontario.

During the summer of 1975, 285 white suckers (Catostomus commersoni) from 10 localities in southern Ontario were examined for trypanosomes. Trypanosoma catostomi Daly and DeGiusti, 1971 was found in the blood of 11.6% of the fish examined using the haematocrit centrifugation technique. Infected juvenile fish (33%) and infected adult fish (4%) were found in 6 of 10 locations.     

Effect of N-ethyl-N-butyl-nitrosamine on the esophageal mucosa of the rat. Histometric investigation of early tumor stages

80 male Wistar rats received an oral ad libitum application of N-ethyl-N-butyl-nitrosamine in a concentration of 0.18 g per litre of drinking water. The changes induced in the esophageal mucosa and determined at three intervals (up to 48 days, up to 91 days, and up to 112 days after commencement of carcinogen exposure) were compared by microscopy with the results from a control group of 10 male Wistar rats of the same age. Several histomorphometric parameters were investigated with the aid of a Leitz ocular micrometer. The earliest localized changes found were an increase in the thickness of the epithelium and the horny layer, and an elongation of the papillary bodies and a widening of the parabasal cellular layer. Later--with a substantial increase in the rate of mitosis in all layers of the epithelium, there was a significant thickening of the non-papillomatous and papillomatous epithelium, an enlargement of the nuclei, especially in the middle and upper layers of the epithelium and a thickening of the horny layer, parts of the latter being parakeratotic. The papillomatous changes corresponded in some cases to moderate epithelial dysplasias. As expected, no fully-developed invasive carcinomas occurred in the early period investigated. The histometric data permit the conclusion to be drawn that the lesions described are demonstrable not only at the exophytic-papillomatous epithelium but also in multifocally localized form at the flat, non-papillomatous mucosa, and that they can definitely be regarded as the expression of an incipient field cancerification.     

Prevalence and intensity ofCapillaria catostomi (Nematoda: Trichuroidea) in white sucker (Catostomus commersoni) in southern Lake Huron,Canada

Synopsis A total of 314 white suckers (Catostomus commersoni) taken monthly during May to December 1975 and in April and May 1976 from southern Lake Huron, Ontario, Canada, were examined forCapillaria catostomi.Prevalence determined from fresh white suckers (66%) was significantly lower than from frozen white suckers (81%). However, there was no significant difference in prevalence ofC. catostomi in samples taken at monthly intervals; in male and female white suckers; or in the age categories.Mean intensity of infection was not significantly different in fresh (9.6 [± 13.41]) or frozen (10.4 [± 13.11]) white suckers. There was no significant difference in intensities between monthly samples in male and female white suckers or in the age categories. Suckers were infected with 1–172 worms. The percentage of male, female and immature worms did not change with sex or age of host or by month. The frequency distribution showed the parasites were overdispersed.A possible life cycle forC. catostomi involving an oligochaete intermediate host is discussed and an attempt made to explain the almost constant levels of prevalence and intensity.     

A study of two species of fish inoculated with spruce budworm nuclear polyhedrosis virus.

Preliminary studies conducted on rainbow trout (Salmo gairdneri) and white suckers (Castostomus commersoni) exposed to spruce budworm nuclear polyhedrosis virus revealed minor histopathologic changes in various organs of each fish which were interpreted as not being significant. However, an evaluation (mean values) of the total changes in groups of fish suggested that a relationship may exist in the suckers exposed to either purified polyhedra or virions. A more extensive and definitive study must be done before any conclusions are warranted.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.     

Ornithine decarboxylase activity and actin polymerization in testosterone--stimulated mouse kidney

Renal hypertrophy was induced in mice by injection of testosterone. Associated with an increase in renal tissue mass were increases in the concentrations of spermine and spermidine and in the activity of ornithine decarboxylase (ODC), the rate limiting enzyme in their synthesis. The increased activity of ODC was shown to be due to an increase in tissue ODC content. Total tissue actin was not altered by testosterone treatment although the proportion of unpolymerized (soluble) actin was increased significantly. These data are discussed in relation to the postulated mechanism of polyamine stimulation of actin polymerization.     

Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.     

ODC-polyamine system is involved in morphine analgesia

alpha-Difluoromethylornithine (DFMO) directly infused into a brain-lateral ventricle (12.5, 25 and 50 micrograms/rat) dose- and time-dependently inhibited brain ODC activity. While having no influence per se on pain threshold, DFMO significantly inhibited the analgesic activity of morphine (15 mg/kg i.p.), this effect being obtained when brain ODC activity was reduced by at least 80%. On the other hand, DFMO had no influence on number and affinity of brain opiate binding sites. Morphine per se neither modified whole brain ODC activity nor significantly affected the ODC inhibitory effect of DFMO. In more discrete brain areas (midbrain, brainstem) morphine actually increased ODC activity. The present results indicate that brain ODC/polyamines system may play a role in the analgesic activity of opioids, probably at a post-receptorial level or through a non-opiate receptor-linked mechanism.