Annamaria Nicolli

Two modes of activation of the permeability transition pore: The role of mitochondrial cyclophilin

Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CsA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-ds-rrafts-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M. (Mol Cell Biochem 174: 181–184,1997)

Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3α/β and Bax, leading to permeability transition pore opening and tumor cell death.

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/β and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/β and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.

Perspectives on the mitochondrial permeability transition

Abstract The permeability transition, a sudden permeability increase of the inner mitochondrial membrane that is greatly favored by Ca2+ accumulation, has puzzled mitochondrial scientists for more than 40 years. It is now recognized that this phenomenon is mediated by opening a high conductance channel (the mitochondrial permeability transition pore) whose open-closed transitions are highly regulated. Through the pore mitochondria may participate in intracellular signalling, and release proteins involved in amplification of the cell death cascade triggered by a variety of physiological and pathological stimuli. Yet, the basic questions of the molecular nature of the permeability transition pore, its physiological role and its very occurrence in vivo remain a matter of intense debate. This short review is meant to summarize our current views on the mitochondrial permeability transition, its perspectives, and our strategies to resolve at least some of the outstanding issues about its nature and function.

Hepatitis B vaccination of adolescents: Significance of non-protective antibodies

Despite hepatitis B virus (HBV) immunization, a percentage of healthy individuals display an antibody titre below the threshold for clinical protection (10 IU/L). In order to predict the existence of this inducible immunological response, the precise anti-HBs titre required to achieve protection in immunized patients with waned HBs antibodies must first be determined. A total of 4486 vaccinated students attending the University of Padova Medical, Science, and Veterinary School were recruited for study between 2004 and early 2012. The baseline concentration of anti-HBs was measured at enrolment. Participants displaying anti-HBs titre < 10 IU/L at the follow-up examination (mean 10.8 years) were given a booster vaccination and retested 20-30 days later. At enrolment, 87.6% of the 4486 vaccinated subjects showed persistence of anti-HBs higher than 10 IU/L. Of the 279 booster-vaccinated subjects, 94.6% achieved the cut-off titre. Booster-induced immunological response was correlated to the pre-booster titre level, with ≥ 2 IU/L ensuring a robust positive response and less than 2 IU/L being associated with the probability of developing insufficient levels of antibodies. Pre-booster antibody titre higher than 2 IU/L in adults might be predictive of an anamnestic response to booster vaccination, whereas titres below this value may indicate likelihood of non-response.

Hepatitis B vaccination at three months of age: A successful strategy?

Vaccination of infants, children and adolescents against the hepatitis B virus (HBV) is mandatory in Italy. It is crucial to assess whether vaccinated subjects have protective antibody level during adulthood when the risk of HBV infection increases due to lifestyle or occupational exposure. Two groups of students attending to University of Padova Medical School were enrolled between 2004 and 2011 and HBV antibodies and antigens were measured. The first group (Group A) comprised students vaccinated at three months of age and the second group (Group B) comprised students vaccinated after the first year of life. The follow-up was 18.0 (Group A) and 17.9 (Group B) years. The students vaccinated at three months of age had a higher rate of non-protective antibodies (47.2%) comparing to those vaccinated after the first year of life (17.0%, P<0.0001) with a significantly lower antibody level (P<0.001). The rate of non-protective antibodies was inversely related to vaccination age. The results clearly show that children vaccinated after the first year of life are better protected against HBV. On the other hand, both groups show a good immunological memory as evidenced by the achievement of protective antibody level after the booster dose in 97.8% of subjects.

Are rats the appropriate experimental model to understand age-related renal drug metabolism and toxicity?

For many years, toxicological investigations have shown that the sensitivity of kidney to xenobiotics evolves depending on the stage of life. The increasing requirement for information on the potential nephrotoxic effect of drugs during human embryonic development, childhood, adulthood and senescence has potentiated toxicological studies in vivo. Rodents, specifically rats, are the primary animal models used in toxicology testing. Despite the popularity of this approach, there are a number of doubts about the appropriateness of rats for the examination of changes in toxicological responses during different stages of life. This perspective tackles the issue of evaluating whether rats fail to adequately mimic the human kidney response to xenobiotic agents through a critical analysis of the literature. We conclude that rats constitute a good model for toxicological investigations during embryonic development, youth and adulthood. However, senescent rats frequently undergo spontaneous kidney degeneration caused by chronic progressive nephropathy, making them a poor model for the study of kidney responses to xenobiotics.

Predictivity and fate of metal ion release from metal-on-metal total hip prostheses.

Blood metal ion levels in 72 patients with large head metal-on-metal hip arthroplasty were studied to determine the correlation between the values measured in whole blood and urine. Urinary cobalt and chromium levels of 30μg and 21μg, respectively, adjusted to creatinine were found to correspond to the 7μg/l cut-off value that has been accepted in whole blood. Cobalt and chromium levels in whole blood and urine both significantly correlated with increased acetabular component inclination angle over 50 degrees and pain scores. There was no correlation with socket anteversion angle or femoral head diameter. The data support the use of urinary measurement of metal ions adjusted to creatinine to monitor patients with large head metal-on-metal total hip arthroplasty.

A simple in vitro test to monitor trace metal toxicity in aqueous samples

Abstract We have studied the effect of test toxicants on electron transfer along the rotenone‐insensitive NADH‐cytochrome b5 reductase of the outer mitochondrial membrane, using oxidized cytochrome c as the electron acceptor in a simple spectrophotometric assay. We show that this reaction is extremely sensitive to trace metals of environmental concern, detecting reliably as little as 10 nM Hg2+. The assay is reproducible, simple, fast and inexpensive, compares favourably with fish acute toxicity tests, and may become useful in the routine monitoring of waters for the presence of this class of pollutants.

Reliability of urinary excretion rate adjustment in measurements of hippuric acid in urine.

The urinary excretion rate is calculated based on short-term, defined time sample collections with a known sample mass, and this measurement can be used to remove the variability in urine concentrations due to urine dilution. Adjustment to the urinary excretion rate of hippuric acid was evaluated in 31 healthy volunteers (14 males and 17 females). Urine was collected as short-term or spot samples and tested for specific gravity, creatinine and hippuric acid. Hippuric acid values were unadjusted or adjusted to measurements of specific gravity, creatinine or urinary excretion rate. Hippuric acid levels were partially independent of urinary volume and urinary flow rate, in contrast to specific gravity and creatinine, which were both highly dependent on the hippuric acid level. Accordingly, hippuric acid was independent on urinary specific gravity and creatinine excretion. Unadjusted and adjusted values for specific gravity or creatinine were generally closely correlated, especially in spot samples. Values adjusted to the urinary excretion rate appeared well correlated to those unadjusted and adjusted to specific gravity or creatinine values. Thus, adjustment of crude hippuric acid values to the urinary excretion rate is a valid procedure but is difficult to apply in the field of occupational medicine and does not improve the information derived from values determined in spot urine samples, either unadjusted or adjusted to specific gravity and creatinine.

Testosterone and Estradiol Affect Renal Oxidative Metabolism and Glutathione Pathway of Wistar Rats

The impact of sexual hormones on kidney metabolism may be relevant to modulate the effect of nephrotoxic substances. In the present research the effects of castration, testosterone, and estradiol on renal oxidative metabolism [cytochrome P-450 (CYP)], glutathione pathway [glutathione content (GSH), glutathione S-transferases (GST) activities, and cysteine-conjugate s-lyase [as glutamine transaminase K (GTK) activity] have been studied. Naive male rats have a significantly lower GSH content but show a significantly higher GST activities and CYP content than females, whereas no sex difference was for GTK activity. Castration significantly reduces GSH content and GTK activity in females and CYP content in males, partially but significantly restored by testosterone. Testosterone significantly increases GSH content and GTK activity in males and GTK activity and CYP content in females. Estradiol increases GSH content in males, whereas decreases GST activities in females and CYP content in both sexes. Castration followed by testosterone treatment increases GTK activity in both sexes whereas increases CYP content and reduces GSH content in females. Castration followed by estradiol treatment increases GSH content and reduces GST activities in males and CYP content in both sexes. In conclusion, the results suggest that sex hormones influence metabolic pathways of the kidney and that they are probably responsible of the sex- related differences of chemical-induced nephrotoxicity. An univocal model to define sex-related toxicity of xenobiotic substances is far to be identified.