Andrea Trevisan

Synthesis of a new platinum(II) complex: anticancer activity and nephrotoxicity in vitro.

New mixed dithiocarbamate-amino Pt(II) complex ([Pt(ESDT)(Py)Cl]) has been recently synthesised with the aim to produce potential anticancer drug able to conjugate cytostatic activity with lack of nephrotoxicity. This complex contains: (1) an amino ligand; (2) a good leaving group (halide); and (3) an S-containing chelating agent potentially able to protect the metal centre from its interaction with S-containing protein-legating sites that are believed to be at the basis of the nephrotoxicity of Pt(II)-based drugs. This complex has been found to be effective as an antiproliferative agent (more active than cis-platin) towards a normal human adenocarcinoma cell line and the corresponding cis-platin-resistant C13 strain. Toxicity tests on the kidney were performed by means of a renal cortical slice model. The slices, prepared with a Brendel-Vitron slicer, were incubated with different doses (0.125-5.0 x 10(-4) M, final concentration) of [Pt(ESDT)(Py)Cl] or cis-platin dissolved in methyl sulphoxide. The platinum(II) complex showed very low renal cytotoxicity as compared with cis-platin; in particular, lipid peroxidation induced by cis-platin appeared about five-fold higher than that induced by [Pt(ESDT)(Py)Cl]. In conclusion, besides being less toxic for the kidney, the results showed that the new synthesised platinum(II) complex appeared in vitro more effective than cis-platin when tested on sensitive and resistant cis-platin tumour cell lines.

Gold(III)-dithiocarbamato anticancer agents: activity, toxicology and histopathological studies in rodents.

Gold(III)‐dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth–inhibitory effects, generally achieved by exploiting non‐cisplatin‐like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum‐based drugs. In this context, [AuIIIBr2(ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin. As a subsequent extension of our previous work, we here report on detailed in vivo studies in rodents, including antitumor activity toward three transplantable murine tumor models, toxicity, nephrotoxicity and histopathological investigations. Remarkably, the gold(III) complex AUL12 stands out for higher anticancer activity than cisplatin toward all the murine tumor models examined, inducing up to 80% inhibition of tumor growth. In addition, it shows low acute toxicity levels (lethal dose, LD50 = 30 mg kg−1) and reduced nephrotoxicity. Altogether, these results confirm the reliability of our drug design strategy and support the validation of this gold(III)‐dithiocarbamato derivative as a suitable candidate for clinical trials.

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.

Renal Proximal Tubule Segment-Specific Nephrotoxicity: An Overview on Biomarkers and Histopathology

The correspondence between histopathological findings and segment-specific biomarkers was investigated in rats treated with segment-specific nephrotoxicants. Male Wistar rats were treated with a single injection of K2Cr2O7 (25 mg/kg sc in saline), cis-Pt (10 mg/kg ip in buffered MSO) or HCBD (100 mg/kg ip in corn oil). Twenty-four and 48 hours after treatment, the rats were sacrificed and the kidneys were drawn for histopathological and biochemical evaluation, i.e., GS activity in renal cortex and PAH uptake in renal cortical slices. Histopathological findings show that cis-Pt and HCBD cause diffuse necrosis of S3 segment of proximal tubules in the outer stripe of outer medulla, respectively. On the contrary, K2Cr2O7 damages exclusively S1–S2 segments, inducing vacuolization at 24 hr and diffuse necrosis at 48 hr after treatment. GS activity in renal tissue is significantly decreased after HCBD and cis-Pt, but not K2Cr2O7 treatment. In contrast, PAH uptake is significantly reduced by K2Cr2O7, but not by cis-Pt or HCBD treatment (even if HCBD causes a slight decrease 48 hr after treatment). The evidence of this study confirms the high specificity of GS activity as marker of S3 segment injury, that PAH uptake is prevalently active in the S1–S2 segments, and that there is complete correspondence among segment-specific nephrotoxicants, biomarkers of segment-specific damage, and histopathological findings.

Kidney injury molecule-1 expression in rat proximal tubule after treatment with segment-specific nephrotoxicants: a tool for early screening of potential kidney toxicity.

Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S3 segment-specific), potassium dichromate (chromate, S1-S2 segment-specific), and cephaloridine (Cph, S2 segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.

Immunity Against Infectious Diseases: Predictive Value of Self-Reported History of Vaccination and Disease

OBJECTIVE To determine whether self-reported history of disease and/or vaccination is predictive of immunity against hepatitis B, varicella, rubella, mumps, and measles. DESIGN The seroprevalence of viral antibodies and the predictive value of a self-report questionnaire were determined for 616 paramedical students who matriculated into Padua Medical School (Padua, Italy) during 2003-2005. RESULTS The majority of subjects (86.9%) remembered being vaccinated against hepatitis B but had no recollection of disease. Among vaccinees, 1.5% showed markers of previous infection, 6.7% tested negative for anti-hepatitis B virus surface antigen (anti-HBsAg) antibodies, and 91.8% tested positive for anti-HBsAg. Self-reported vaccination history had a positive predictive value of 93.2% for test results positive for immunity against hepatitis B. Immunity against varicella (93.7% of subjects) and rubella (95.5%) was high, compared with immunity against mumps (79.9%) and measles (83.1%). In addition, results of tests for detection of immunity against mumps and measles were equivocal for more than 7% of subjects, probably because their vaccination regimen was not completed. Self-reported histories of varicella disease and rubella disease and vaccination had high positive predictive values (greater than 98% each) for testing positive for antiviral antibodies, compared with self-reported histories of mumps disease and vaccination and measles disease and vaccination; however, high positive predictive values were observed for self-reported histories of mumps only (92.0%) and measles only (94.7%). CONCLUSIONS The self-report questionnaire used in this study did not accurately predict immunity against 5 transmittable but vaccine-preventable diseases. A complete serological evaluation of healthcare workers, followed by vaccination of those with negative or equivocal results of serological tests, is an appropriate measure to decrease the risk of infection in this population.

Glutamine synthetase activity in rat urine as sensitive marker to detect S3 segment-specific injury of proximal tubule induced by xenobiotics.

Abstract The possibility of detecting segment-specific injury of the proximal tubule by means of urinary enzymes was investigated in rats. Urinary glutamine synthetase, an enzyme exclusively localized in the S3 segment, and N-acetyl-β-d-glucosaminidase, prevalently a S1-S2, but S3 enzyme also, were determined after single treatment with 100 mg/kg body wt. of hexachloro-1:3-butadiene (HCBD; i.p.), toxic for the S3 segment, or 25 mg/kg body wt. of potassium dichromate (s.c.), toxic for the S1-S2 segments. Excretion of total urinary proteins was also measured. In addition, a dose-response relationship was determined between three doses (50, 100 and 200 mg/kg body wt.) of HCBD and glutamine synthetase activity in urine. Glutamine synthetase activity, measured according to a new assay for urine based on modification of methods developed for organs, increased in the urine only when the S3 segment of the proximal tubule was damaged, as demonstrated by histological findings of the kidneys. HCBD caused early excretion of the enzyme related to the necrosis of the S3 segment, whereas potassium dichromate caused a slight increase only when the resulting lesion to this segment (vacuolization) began to develop. On the contrary, N-acetyl-β-d-glucosaminidase activity showed the peak of excretion 24 and 34 h after treatment with HCBD or potassium dichromate, respectively, according to the histological findings of necrosis of the S3 segment (the former) and vacuolization of the S1-S2 segments (the latter). Excretion of total urinary proteins reached the peak 24 h (HCBD) and 48 h (potassium dichromate) after treatment. HCBD at 200 mg/kg body wt. caused a peak of glutamine synthetase activity in urine 10 h after injection, whereas the peak caused by doses of 50 and 100 mg/kg body wt. occurred 24 h following treatment. The peak of enzyme activity in urine significantly increased with the dose. The results suggest that the measurement of urinary activity of S3 segment-specific enzyme as glutamine synthetase allows us to detect early S3 segment-specific injury of the proximal tubule. In addition, the method for urinary enzyme activity appears sensitive, simple and fast.

Synthesis of a palladium(II)-dithiocarbamate complex: biological assay and nephrotoxicity in rats

Abstract. A new palladium(II)-dithiocarbamate complex, [Pd(ESDT)Cl]n, was synthesised and its chemical characteristics are discussed. This complex was examined for its cytotoxic properties in human tumour cell lines; for comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions. In particular, Pd(II)-complex cytotoxicity on ovarian carcinoma C13 cells, resistant to cisplatin, showed that there seemed to be no cross-resistance between [Pd(ESDT)Cl]n and cisplatin. The effects on the kidney were also studied. Biochemical investigation on urinary parameters showed that the effects after a single injection are similar to those of cisplatin, with an increase of urinary proteins and enzyme excretion in urine, and a significant decrease of glutamine synthetase activity in the renal tissue. In addition, the Pd(II)-complex caused a significant decrease of p-aminohippuric acid uptake in renal cortical slices relative to cisplatin. On the other hand, histopathological findings showed that the effects of the Pd(II)-complex are more severe and diffuse than the damage caused by cisplatin. Biochemical and histopathological findings show that the Pd(II)-complex affects the pars recta and pars convoluta, in contrast to cisplatin, which only affects the pars recta.

Biological monitoring of cadmium exposure: reliability of spot urine samples

SummaryConcentration-dilution of spot urine samples is a shortcoming of the biological monitoring of industrial xenobiotics. To ascertain whether the adjustment of urinary cadmium measured in spot samples is appropriate, urine samples were taken three times, once a week for 3 successive weeks, from 25 welders employed in the manufacture of jewellery (total 75 samples). Cadmium, creatinine, specific gravity, total urinary solutes, urinary volume and urinary flow rate were measured in 12-h collections and in spot samples taken immediately afterwards. Creatinine and total urinary solutes showed high inverse correlation with urinary flow rate (r = −0.858 and r = −0.768 respectively). Urinary cadmium displayed a similar trend but the correlation was not significant (r = −0.145). Creatinine adjustment of urinary cadmium values in spot samples increased the correlation with the same index in timed samples adjusted for urinary volume (r = −0.808) or urinary flow rate (r = 0.821) compared with non-adjustment (r = 0.732 and r = 0.738, respectively). Creatinine adjustment of spot sample values is also suitable for a wide range of urinary concentrations; discarding excessively diluted or concentrated urines, correlation of urine samples improved for non-adjusted or specific gravity-adjusted values, whereas no changes were observed for creatinine-adjusted values.

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.