Biochemical and cytogenetic biomarkers of pollutant exposure in marine fish (Aldrichetta forsteri Valenciennes and Sillago schomburgkii Peters) near industrial and metropolitan centres in South Australia

This project aimed to measure biochemical and cytogenetic biomarkers in marine fish (Aldrichetta forsteri and Sillago schomburgkii) associated with industrial and urban centres in South Australia. These sites were Port Pirie (affected by metal-contaminated outflows), Barker Inlet (adjacent to Metropolitan Adelaide), and Wills Creek (reference site). The biochemical biomarkers included sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) in serum, adenylate levels (ATP, ADP and AMP) and adenylate energy charge (AEC) in gill and liver, and sodium/potassium ATPase (Na⁺, K⁺-ATPase) in gill. Erythrocyte micronucleus frequency was a marker of cytogenetic effect. Serum enzyme levels were generally higher in fish from Port Pirie and Barker Inlet than in those from Wills Creek, with SDH demonstrating the clearest site-associated differences. Tissue adenylates were consistently lower at Port Pirie than elsewhere, suggesting a greater metabolic strain in fish at this site. AEC in gill and liver were consistently lower at Port Pirie than at Wills Creek, with Barker Inlet generally between these two. The reversed rank order was observed with erythrocyte micronucleus frequencies. Seasonal variations in the biomarkers may be attributed either to seasonal physiological changes in fish or changes in pollutant input levels or compositions. Na⁺, K⁺-ATPase did not differ between sites nor seasons in this study. This work shows that biochemical and cytogenetic differences occur in marine fish at specific locations in South Australia. It also shows that of these tests, serum SDH and erythrocyte micronuclei are potentially the most sensitive and reliable biomarkers of pollutants effects on marine fish. The results also suggest that these data may be used as a baseline against which future changes in marine water quality, and their consequent biological effects, can be compared.     

Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.     

An open issue: the inner mitochondrial membrane (IMM) as a free boundary problem

The inner mitochondrial membrane (IMM) is structured in cristae, which contributes to the best functioning of ions and adenylates exchange between the matrix and the intermembrane space. The central hypothesis of this paper is that the cristae structure favours a minimal mean free path of adenylates between translocation sites (translocase/ANT sites) and metabolic sites (ATPase sites). We propose a mathematical model and then give simulations. Based on simple hypotheses about cristae growth, they show that we can account for the major features of the IMM organization and functioning by minimizing the mean interdistance between ADP/ATP translocation and transformation sites.     

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37–38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.     

Possible role of adenylates in the engagement of the cyanideresistant pathway in nutrient-starved Catharanthus roseus Cells

In Cathuranthus roseus (L.) G. Don cells the cyanide-resistant pathway is engaged after phosphate or nitrogen starvation. Re-addition of these nutrients disengaged it again. Re-addition of phosphate leads to a transient disengagement which becomes only permanent after a second addition of phosphate. Disengagement after re-addition of nitrogen is slow: it takes 9 days before the activity has disappeared. In this system the mechanism of engagement of the cyanide-resistant pathway was studied. Addition of phosphate to phosphate-starved cells induced cell division within 24 h. The disengagement of the cyanide-resistant pathway was probably only an indirect effect of phosphate because the cellular P, content, which increased rapidly after addition, was low again before the cyanide-resistant pathway was disengaged. A better correlation was observed between high ADP and adenylate content of the cells and disengagement of the cyanide-resistant pathway. In addition it appeared that the engagement of the cyanide-resistant pathway was not the result of a limited carrier capacity of the cytochrome pathway. It is tentatively concluded that the engagement of the cyanide-resistant pathway in phosphate-starved cells was the result of a limited adenylate content. After nitrogen addition to N-starved cells, it took 5 days until the first growth occurred. Before the cyanide-resistant pathway was disengaged, its activity increased with the increased respiration rate which preceded growth. Within 72 h a higher ADP content was observed, which was still high after 10 days. The stimulation of the cytochrome pathway by uncoupler was small and more or less the same with and without added nitrogen, as long as the cyanide-resistant pathway was engaged. After disengagement the stimulation by uncoupler was significantly larger. It is suggested that the engagement during N-starvation was the result of a limited carrier capacity of the cytochrome pathway. Stimulation of the metabolism by re-addition of phosphate, nitrogen or sucrose resulted in a rapid increase in the levels of uracil nucleotides and uridine diphosphoglucose (UDPG) which are involved in sucrose metabolism.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Biochemical and cytogenetic biomarkers of pollutant exposure in marine fish (Aldrichetta forsteri Valenciennes and Sillago schomburgkii Peters) near industrial and metropolitan centres in South Australia

This project aimed to measure biochemical and cytogenetic biomarkers in marine fish (Aldrichetta forsteri and Sillago schomburgkii) associated with industrial and urban centres in South Australia. These sites were Port Pirie (affected by metal-contaminated outflows), Barker Inlet (adjacent to Metropolitan Adelaide), and Wills Creek (reference site). The biochemical biomarkers included sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) in serum, adenylate levels (ATP, ADP and AMP) and adenylate energy charge (AEC) in gill and liver, and sodium/potassium ATPase (Na⁺, K⁺-ATPase) in gill. Erythrocyte micronucleus frequency was a marker of cytogenetic effect. Serum enzyme levels were generally higher in fish from Port Pirie and Barker Inlet than in those from Wills Creek, with SDH demonstrating the clearest site-associated differences. Tissue adenylates were consistently lower at Port Pirie than elsewhere, suggesting a greater metabolic strain in fish at this site. AEC in gill and liver were consistently lower at Port Pirie than at Wills Creek, with Barker Inlet generally between these two. The reversed rank order was observed with erythrocyte micronucleus frequencies. Seasonal variations in the biomarkers may be attributed either to seasonal physiological changes in fish or changes in pollutant input levels or compositions. Na⁺, K⁺-ATPase did not differ between sites nor seasons in this study. This work shows that biochemical and cytogenetic differences occur in marine fish at specific locations in South Australia. It also shows that of these tests, serum SDH and erythrocyte micronuclei are potentially the most sensitive and reliable biomarkers of pollutants effects on marine fish. The results also suggest that these data may be used as a baseline against which future changes in marine water quality, and their consequent biological effects, can be compared.     

Respiratory and metabolic responses of the Australian Yabby Cherax destructor to progressive and sustained environmental hypoxia

The Australian Yabby, Cherax destructor, inhabits occasionally hypoxic water. The respiratory gas, acid-base, metabolite and energetic status of this crayfish was assessed during progressive hypoxia and during 3 h at a water PO₂ of 1.33 kPa. The O₂ affinity of haemocyanin from C. destructor was increased by lactate (Δlog P ₅₀/Δlog[lactate] = −0.111) and by Ca (Δlog P ₅₀/Δlog[Ca] = −0.62) but not by urate. While the non-bicarbonate buffering capacity was low (Δ[HCO₃ ⁻]/ ΔpH=−4.89) the haemocyanin had a low sensitivity to pH changes (ϕ = −0.33). The crayfish showed a compensatory hyperventilation, which induced a respiratory alkalosis, until the water O₂ partial pressure declined below 2.67 kPa, after which the O₂ uptake rate was approximately 10% of normoxic rates. The high haemocyanin-O₂ affinity maintained haemolymph O₂ content during progressive hypoxia despite the normally low arterial O₂ partial pressure of C. destructor. During severe hypoxia, pH decreased but increased lactate aided in maintaining haemocyanin-O₂ saturation. The importance of regulated haemocyanin-O₂ affinity in hypoxic C. destructor was reduced by lowered metabolism, including reduced cardiac output, and the consequent reduction in O₂ requirement. Anaerobiosis became important only at very low PO₂ but thereafter proceeded rapidly, supported by a marked hyperglycaemia. There was no depletion of adenylates, even after 3 h of severe hypoxia. The tail muscle of C. destructor held small amounts of glycogen which would sustain anaerobiosis for a only a few hours. Hypometabolism seems an important hypoxic response but severe hypoxia may encourage the crayfish to breathe air. Accepted: 26 February 1998     

Changes in ATP in relation to floral induction and initiation in Pharbitis nil

No changes in metabolism of adenosine phosphates as a function of short day induction were detected in cotyledons of Pharbitis nil Chois strain Violet. A gradual increase in ATP level was detected throughout the dark period in plumules. A rapid decline of ATP pool size was observed in induced plumules shortly after floral induction. The decline occurred close to the 14th hour of the dark period, 1 to 1.5 h after the dark period length required for a 90% flowering response, which is thought to be the minimum time required for transport of the floral stimulus (and assimilates) from the induced cotyledons to the plumule. Transport of the major adenylates from the cotyledons was verified using [¹⁴C]-adenine. Estimates of the amount, and rate, of adenylate transport suggest that the cotyledons could be an important source of adenylates to re-establish the ATP pool size in evoked plumules.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3.I. Effects of nitrogen Limitation

The growth rate of Lemna giba L. G3 was varied by limiting the supply of nitrogen (N) under otherwise constant condition. Two experimental approaches were used. 1) A series of suboptimally growing cultures were supplid daily with exponentially increasing doses on N. 2) Optimally growing cultures were transferred to a N-fre medium and cultivated in it for 10 days. Lveles of starch, sucrose, glucose, fructose, and systems. At RGR ranging from optimal to 50% of optimal caused decreased levels of soluble sugars, but increased lvels of starch.Starch accumulation showed a strong negative correlation with the CO² assimilation rate, indicating increased triose phosphate/inorganic phosphate (TP/Pⁱ) ratio in the chloroplast causing end product inhibition of photosyntheisis. The data indicat the quantitative rlationship betwen the photosynthetic activity and the carbon utilization rate influnces the activity of the sucrose synthesis pathway and thus the rate of the triose hosphate/Pⁱ exchange at the chloroplast membrane, which in turn regulates the activity of starch synthesis and the Calvin cycle.