TRC-PAD: Accelerating Recruitment of AD Clinical Trials through Innovative Information Technology.

BACKGROUND: The Trial-Ready Cohort for Preclinical/Prodromal Alzheimer's Disease (TRC-PAD) Informatics Platform (TRC-PAD IP) was developed to facilitate the efficient selection, recruitment, and assessment of study participants in support of the TRC-PAD program. OBJECTIVES: Describe the innovative architecture, workflows, and components of the TRC-PAD IP. DESIGN: The TRC-PAD IP was conceived as a secure, scalable, multi-tiered information management platform designed to facilitate high-throughput, cost-effective selection, recruitment, and assessment of TRC-PAD study participants and to develop a learning algorithm to select amyloid-bearing participants to participate in trials of early-stage Alzheimer's disease. SETTING: TRC-PAD participants were evaluated using both web-based and in-person assessments to predict their risk of amyloid biomarker abnormalities and eligibility for preclinical and prodromal clinical trials. Participant data were integrated across multiple stages to inform the prediction of amyloid biomarker elevation. PARTICIPANTS: TRC-PAD participants were age 50 and above, with an interest in participating in Alzheimer's research. MEASUREMENTS: TRC-PAD participants' cognitive performance and subjective memory concerns were remotely assessed on a longitudinal basis to predict participant risk of biomarker abnormalities. Those participants determined to be at the highest risk were invited to an in-clinic screening visit for a full battery of clinical and cognitive assessments and amyloid biomarker confirmation using positron emission tomography (PET) or lumbar puncture (LP). RESULTS: The TRC-PAD IP supported growth in recruitment, screening, and enrollment of TRC-PAD participants by leveraging a secure, scalable, cost-effective cloud-based information technology architecture. CONCLUSIONS: The TRC-PAD program and its underlying information management infrastructure, TRC-PAD IP, have demonstrated feasibility concerning the program aims. The flexible and modular design of the TRC-PAD IP will accommodate the introduction of emerging diagnostic technologies.

Effect of litter size on prepartum metabolic and amino acidic profile in rabbit does.

The use of modern prolific lines of rabbit does in intensive production systems leads to an increase in productivity but also causes a rise in several problems related to the does' health status. Hence, the aim of this study was to investigate the effect of the litter size on the metabolic, inflammatory and plasma amino acid profile in rabbit does. The blood of 30 pregnant does was sampled on the 27th day of pregnancy. The does were retrospectively grouped according to the number of offspring into a high litter size group (HI, does with ≥ 12 kits; n = 16) and a low litter size group (LO, does with ≤ 11 kits; n = 14). Data were subjected to Pearson's correlation analysis. Further, data were analysed in agreement to a completely randomized design in which the main tested effect was litter size. The linear or quadratic trends of litter size on parameters of interests were post hoc compared by using orthogonal contrasts. In addition, compared with the LO group, the HI group had lower levels of glucose (-5%; P < 0.01), zinc (-19%; P < 0.05), albumin (-6%; P < 0.05) and total cholesterol (-13%; P < 0.07), but the total bilirubin level was higher in the HI group (+14%; P < 0.05). Regarding the plasma amino acids, the HI group had lower concentrations of threonine (-15%), glycine (-16%), lysine (-16%) and tryptophan (-26%) and a higher level of glutamic acid (+43%; P < 0.05) compared with the LO group. The exclusively ketogenic amount of amino acids was lower (P < 0.06) in the HI (55.8 mg/100 ml) does compared with the LO does (56.8 mg/100 ml). These results show that a few days before delivery, rabbit does that gave birth to a higher number of offspring had a metabolic profile and an inflammatory status that was less favourable with respect to does who gave birth to a lower number of offspring. Moreover, the plasma amino acid profile points out that there was an enhanced catabolic condition in the rabbit does with a high number of gestated foetuses; it was likely related to the greater energy demand needed to support the pregnancy and an early inflammatory response.

Enhancement of stress corrosion cracking of AZ31 magnesium alloy in simulated body fluid thanks to cryogenic machining.

Magnesium and its alloys have recently attracted great attention as potential materials for the manufacture of biodegradable implants. Unfortunately, their inadequate resistance to the simultaneous action of corrosion and mechanical stresses in the human body have hampered their use as implant materials. This work aims at evaluating the Stress Corrosion Cracking (SCC) susceptibility of the AZ31 Mg alloy after being machined under cryogenic cooling. The SCC behaviour was evaluated by means of Slow Strain Rate Tests (SSRTs) in Simulated Body Fluid (SBF) at 37 °C. Prior to testing, a full characterization of the machined surface integrity, including microstructural observations, residual stress, nano-hardness measurements and surface texture analysis was carried out together with the assessment of the corrosion properties through potentiodynamic polarization curves. In addition, the morphology of the fracture surfaces after SSRTs was analysed by means of 3D optical profiler and Scanning Electron Microscopy (SEM). The improved corrosion resistance due to the increased extension of the nano-surface layer and to the compressive residual stresses represents the reason of the reduced SCC susceptibility of cryogenically machined AZ31 samples as compared to dry machined ones.

Technical note: Evaluation of a novel enzymatic method to predict in situ undigested neutral detergent fiber of forages and nonforage fibrous feeds.

The purpose of this study was to optimize the conditions of a previously proposed enzymatic method used to estimate in situ undigested neutral detergent fiber (uNDF). We used a multi-step enzymatic approach, in which samples were first solubilized in NaOH solutions as a preincubation (PreInc) phase. After rinsing, samples were incubated (24 h at 39°C) in a buffered solution (pH 6) containing hemicellulase, cellulase, and Viscozyme L enzymes (Sigma-Aldrich s.r.l., Milan, Italy), followed by incubation (24 h at 39°C) in a buffered solution (pH 5) containing xylanase. Two sets of experiments were performed: a calibration trial (that tested different PreInc conditions on 9 selected forages) and a validation trial (that verified the results by testing multiple samples of 6 different forage types and a group of fibrous by-products). In the calibration trial, samples (300 mg in Ankom F57 filter bags; Ankom Technology Corp., Fairport, NY) were preincubated at 39°C in a 0.1 M NaOH solution for 90, 180, or 240 min, or in 0.2, 0.5, 1.0, or 2.0 M NaOH solution for 90 min. The results indicated that the best PreInc method, in terms of intra-laboratory repeatability and estimation of reference in situ values, was 90 min in a 0.2 M NaOH solution. Thus, we used this PreInc condition to determine enzymatic uNDF of 257 samples in the validation trial. Although the selected method generally had good accuracy in predicting in situ uNDF, inconsistencies were noted for certain forage types. Overall, when enzymatic uNDF was used to predict the in situ uNDF of all samples, the regression was satisfactory (intercept = 7.098, slope = 0.920, R2 = 0.73). The regression models developed for alfalfa hays, corn silages, and small grain silages had also acceptable regression performances and mean square error of prediction (MSEP) values, and the main sources of MSEP variation were error due to incomplete (co)variation and random error. Even when R2 values were >0.70, the MSEP value of the regression model for grass hays was 149.55, and that for nonforage fibrous feeds was 155.16. Although enzymatic uNDF partially overestimated the in situ uNDF, particularly in grass silages, the proposed procedure seems to be promising for accurately predicting in situ uNDF, because it generally had good repeatability and provided satisfactory estimates of in situ uNDF.

Technical note: Relationship between in situ NDF degradability and enzymatic NDF hydrolysis in forages, nonforage fibrous feeds, and crop residues.

The study was performed on forages ( = 8), nonforage fibrous feeds ( = 10), and crop residues ( = 2). Samples were characterized for in situ NDF degradability (NDFD) at 6, 12, 18, 24, 30, 36, 48, 72, 96, 120, and 240 h of ruminal incubation. Then, samples were characterized for enzymatic NDFD by adopting a multistep enzymatic method consisting of a preincubation (PreInc) phase followed by enzymatic incubation (EnzInc) steps. In the PreInc phase, samples were incubated in a NaOH solution for 0, 30, 60, or 90 min. Then, in the EnzInc phase, samples were first incubated in a buffered enzymatic solution containing hemicellulase, cellulase, and Viscozyme L enzymes. Then, samples were incubated in a xylanase-buffered enzymatic solution. These 2-step EnzInc lasted for a total of 16 (8 h for the first enzymatic step + 8 h for the second enzymatic step), 32 (16 + 16 h), or 48 h (24 + 24 h). The enzymatic NDFD coefficients were increased by increasing both PreInc and EnzInc incubation times, and no PreInc × EnzInc interaction was observed, except for ryegrass hay. On average, enzymatic NDFD increased ( < 0.05) by 0.35, 0.54, or 0.68, respectively, for 30-, 60-, or 90-min PreInc compared with 0-min PreInc. The enzymatic NDFD increased ( < 0.05), on average, by 0.11 in 32-h EnzInc or 0.16 in 48-h EnzInc with respect to 16-h EnzInc. Enzymatic NDFD were used to predict in situ NDFD coefficients by adopting single regression equations. High coefficients of regression ( > 0.80, < 0.05) and low errors of prediction were measured when specific enzymatic conditions were performed to predict in situ NDFD at intermediate (from 24 to 48 h) ruminal incubation. Generally, worse regression performances were obtained when enzymatic NDFD were used to predict in situ NDFD evaluated after shorter or longer incubation times. The direct prediction of the rate of NDF degradation was not possible using enzymatic NDFD coefficients. Even if the proposed multistep enzymatic method appeared promising, further studies are required to improve enzymatic NDFD prediction ability within specific forage types or nonforage fibrous feeds.

The "Eyeballing" technique: an emerging and alerting trend of alcohol misuse.

OBJECTIVE: Alternative methods of alcohol consumption have recently emerged among adolescents and young adults, including the alcohol "eyeballing", which consist in the direct pouring of alcoholic substances on the ocular surface epithelium. In a context of drug and behavioural addictions change, "eyeballing" can be seen as one of the latest and potentially highly risky new trends. We aimed to analyze the existing medical literature as well as online material on this emerging trend of alcohol misuse. MATERIALS AND METHODS: Literature on alcohol eyeballing was searched in PsychInfo and Pubmed databases. Results were integrated with a multilingual qualitative assessment of the database provided by The Global Public Health Intelligence Network (GPHIN) and of a range of websites, drug fora and other online resources between March 2013 and July 2013. RESULTS: Alcohol eyeballing is common among adolescents and young adults; substances with high alcohol content, typically vodka, are used for this practice across the EU and internationally. The need for a rapid/intense effect of alcohol, competitiveness, novelty seeking and avoidance of "alcoholic fetor" are the most frequently reported motivations of "eyeballers". Local effects of alcohol eyeballing include pain, burning, blurred vision, conjunctive injection, corneal ulcers or scarring, permanent vision damage and eventually blindness. CONCLUSIONS: Alcohol eyeballing represents a phenomenon with potential permanent adverse consequences, deserving the attention of families and healthcare providers. Health and other professionals should be informed about this alerting trend of misuse. Larger observational studies are warranted to estimate the prevalence, characterize the effects, and identify adequate forms of interventions for this emerging phenomenon.

Positive compared with negative margins in a single-centre retrospective study on 3957 consecutive excisions of basal cell carcinomas. Associated risk factors and preferred surgical management.

The rate of margins involvement and the associated recurrence risk in basal cell carcinomas (BCCs) varies widely in published works (7%-25% and 26%-67%, respectively). This study investigated the risk factors associated with incomplete excision and their relevance in surgical management when positive margins occur in 3957 BCCs excised in 2358 patients. This study performed a multivariate analysis on the database collected from all patients operated for BCCs in the Plastic Surgery Department between 1 January 1992 and 1 September 2007. All data collected (3957 excisions; 2358 individuals) were divided into complete and incomplete excisions groups and analyzed according to 14 variables. The overall rate of incomplete excisions was 14%. Mean age (68), size of the lesion (< 0.5 cm), BCC subtype (nodular with sclerosant aspects, sclerosant and basosquamous), location (face), infiltration depth (hypodermis and deep tissues), recurrent BCC and re-excised BCC were significantly associated with a higher rate of incomplete excision. The recurrence rate for incompletely excised tumours was 26.8%, while only 5.9% for completely excised tumours. Most of the risk factors associated to incomplete excision can be identified before surgery (by simple anamnesis and clinical examination) and successfully overcome by appropriate surgical margins. The high recurrence rate after incomplete excision and the low patient compliance towards follow-up should lead the surgeon to early re-excise residual cancer.

Limb contouring after massive weight loss: functional rather than aesthetic improvement.

BACKGROUND: After massive weight loss, both upper and lower limbs show a similar deformity which consists of redundancy and ptosis of the cutaneous mantle. Many disturbances are associated with this abnormality, which can be treated surgically. A retrospective review of limb-contouring procedures after massive weight loss is presented. METHODS: Thigh lift and arm lift procedures are described. All surgeries of upper and lower limbs contouring performed between 2003 and 2006 are reviewed with regard to quantity of tissue removed, comorbidities, complications and patients' satisfaction, which was surveyed through a questionnaire exploring functional and esthetic results (maximum score 3). RESULTS: Among 48 bilateral limb-contouring procedures, medial thigh lifts were 35 (73%) and brachioplasties were 13 (27%). Mean age was 46 and average body mass index variation was 20 kg/m(2). The most frequent comorbidity was gallstones (28%). In 46% of the whole group of patients, there was no complication to mention. The most frequent complication was acute anaemia in both procedures (43% in thigh lift and 54% in arm lift). Mean quantity of adipose-dermoid tissue removed was 766 g in thigh lift and 463 g in arm lift. In case of surgery combined with liposuction, the average aspirated volume was 1,933 ml (thighs) and 1,117 ml (arms). Patients' satisfaction was 2.7 for thighs and 2.6 for arms, as average. CONCLUSION: The rate of complications in limb contouring after weight loss is higher than the analogue esthetic procedures. Nevertheless, due to the rehabilitative significance of limb surgery after weight loss, this step is to be included as fundamental in obese patients' surgical therapy.

Delayed treatment of persistent radial nerve paralysis associated with fractures of the middle third of humerus: review and evaluation of the long-term results of 52 cases.

This study was undertaken to determine the efficacy of delayed surgical treatment in cases of persistent radial nerve paralysis after fractures of the middle third of the humerus. We have limited this study to patients who had absolutely no functional recovery of the radial nerve 3 to 4 months after middle third humeral fractures. The fractures were treated by a variety of orthopaedic methods, conservative and surgical, in other departments and hospitals. Surgical exploration of the radial nerve was carried out 3 to 4 months after primary orthopaedic treatment. The outcome of this study concurs with data in the literature in showing that delayed nerve surgery (neurolysis or nerve grafts) in the absence of functional recovery of the radial nerve after humeral fracture can be useful in achieving good functional recovery and subjectively satisfying results.

Co-purification of mitochondrial HSP70 and mature protein disulfide isomerase with a functional rat kidney high-M(r) cysteine S-conjugate beta-lyase.

S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC, the cysteine S-conjugate of tetrafluoroethylene) is an example of a nephrotoxic, halogenated cysteine S-conjugate. Toxicity results in part from the cysteine S-conjugate beta-lyase(s)-catalyzed conversion of TFEC to a thioacylating fragment with the associated production of pyruvate and ammonia. In the present study, we have demonstrated that rat kidney homogenates contain at least three enzyme fractions that are capable of catalyzing a cysteine S-conjugate beta-lyase reaction with TFEC. One of these fractions contains a high-M(r) lyase. At least two proteins co-purify with this high-M(r) complex. N-Terminal analysis (15 cycles) revealed that the smaller species was mature protein disulfide isomerase (M(r) approximately 54,200) from which the 24 amino acid endoplasmic reticulum signal peptide had been removed. Internal amino acid sequencing (15 cycles) revealed that the larger species was mitochondrial HSP70 (mtHSP70; M(r) approximately 75,000). The present findings offer an explanation for the previous observation that mtHSP70 in kidney mitochondria is heavily thioacylated when rats are injected with TFEC (Bruschi et al., J Biol Chem 1993;268:23157-61).

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

The distally based superficial sural flap: our experience in reconstructing the lower leg and foot.

The treatment of soft-tissue defects of the lower third of the leg and foot is often an awkward problem to tackle because of the frequent involvement of muscle, tendon, and bone, which is caused by the thinness and poor circulation of the skin covering them and by the small quantity of local tissue available for reconstruction. The authors present their experience with the use of sural flaps for the treatment of small- and medium-size defects of the distal region of the lower limb. The flap used was a distally based fasciocutaneous flap raised in the posterior region of the lower two thirds of the leg. Vascularization was ensured by the superficial sural artery, which accompanies the sural nerve together with the short saphenous vein. The authors treated 18 patients (12 men and 6 women) from May 1997 to August 1999 at the Division of Plastic Surgery, University of Turin, Italy. Superficial necrosis without involvement of the deep fascia (which was grafted 1 month later) occurred in 1 patient of the 18 treated. In another 2 patients, defects were found in the flap margins, but no additional surgical revision was necessary, and recovery occurred by secondary intention. In every patient the sural flaps provided good coverage of the defects, both from a functional and an aesthetic point of view. The major advantages of this flap are its easy and quick dissection. Because the major arterial axis is not sacrificed, this flap can be used in a traumatic leg with damaged major arteries.

Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death.

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of alphaKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of alphaKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.

[Cranioplasty with polymethylmethacrylate. The clinico-statistical considerations]. / Cranioplastica con polimetilmetacrilato. Considerazioni clinico-statistiche.

BACKGROUND: The main purpose of the reconstruction of the cranium is the protection of the brain. Besides we have to consider important functional and aesthetic necessities in order to achieve satisfactory results. METHODS: Thirty-six clinical cases, operated from November 1991 to June 1996, in which the reconstruction of the cranial vault is carried out by a polymethylmethacrylate acrylic resin are analysed. The causes and locations of the most common bone defects and the main indications for reconstruction are examined. While the repair of the osseous gaps caused by neoplasms is immediate, in the traumatic occurrences, in order to reduce the probability of infectious complications, an average time of 11 months elapsed from the first operation. The surgical technique, with slightest alterations, is the same in all the presented cases, preparing the acrylic resin straight on the operating table. The resin, moulded and adapted to the defect until its complete hardening, presents, thanks to its properties, manifold advantages (and few real disadvantages). RESULTS: The results, in terms of complications, are very satisfactory, with an infectious rate of 2.7%. Besides, in one third of the patients, a considerable clinical improvement after the repair has been observed. CONCLUSIONS: According to personal experience, it is possible to affirm that polymethylmethacrylate, with its remarkable plasticity and stability in time, can always guarantee a satisfactory functional and aesthetic result.

Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad. Sci. USA 93, 1671-1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted ove the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, i.p.) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (> 90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 doses not play a significant role in APAP hepatotoxicity in these mice.

Mitochondrial stress protein actions during chemically induced renal proximal tubule cell death.

We have previously shown that the potent mammalian nephrotoxicant tetrafluoroethyl-L-cysteine (TFEC) covalently modifies a select group of mitochondrial proteins prior to cell death. More recently we have identified these adducted proteins as subunits of mitochondrial dehydrogenase multienzyme complexes, which are involved in key regulatory steps of cellular respiration. Most importantly the E2 and E3 subunits of alpha-ketoglutarate dehydrogenase are adducted. We report here that the consequence of adduction is the formation of tertiary complexes between dehydrogenase subunits and the mitochondrial heat shock protein 60 (HSP60) and a HSP70 homolog (mortalin/PBP74). Thus, adduction perturbs protein structural integrity sufficiently to allow for mitochondrial stress protein recognition. These data also suggest that, in our mammalian system, HSP60 appears to act in the identification and maintenance of protein integrity, as has been previously established for simpler eukaryotic systems.

Mitochondrial HSP60 (P1 protein) and a HSP70-like protein (mortalin) are major targets for modification during S-(1,1,2,2-tetrafluoroethyl)-L-cysteine-induced nephrotoxicity.

The potent and site-selective nephrotoxicity of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) in vivo has been associated with difluorothioamidyl-L-lysine formation on critical mitochondrial target proteins. Dose-response studies in the Fischer 344 rat indicate that five proteins with apparent molecular masses of 99, 84, 66, 52, and 48 kDa are predominantly adducted in vivo after nephrotoxic doses of TFEC (> 10 mg/kg, intraperitoneally). Microsequence analysis of the major difluorothioamidyl-L-lysine proteins indicated that P66 is identical, over 14 NH2-terminal residues, to mitochondrial P1 protein (HSP60, a chaperonin) and that P84 is identical, over 14 residues, to a recently isolated novel member of the HSP70 family known as mortalin. These studies indicate that mitochondrial heat shock proteins are major targets for modification by reactive metabolites of TFEC. The implications of these data in relation to the nephrotoxicity of cysteine conjugates are discussed.

In vitro cytotoxicity of mono-, di-, and trichloroacetate and its modulation by hepatic peroxisome proliferation.

Dichloroacetate (DCA) and trichloroacetate (TCA) are major by-products of drinking water chlorination. Recent experiments have shown that both of these compounds produce hepatic tumors in B6C3F1 mice. There was evidence that these effects may be associated with cytotoxic effects and/or peroxisomal proliferation. Therefore, in the present study the in vitro cytotoxicity of monochloroacetate (MCA), DCA, TCA and a metabolite, glycolate (GLY), was determined in hepatocyte suspensions prepared from naive and clofibric acid-pretreated male Sprague-Dawley rats and B6C3F1 mice. Cytotoxic responses, measured by release of lactic dehydrogenase and/or trypan blue exclusion, were only observed with high concentrations (5.0 mM) of MCA and GLY in hepatocytes from naive animals (p = 0.025 and 0.008, respectively, Sprague-Dawley rat; p = 0.033 and 0.001, respectively, B6C3F1 mouse). The cytotoxic responses to both compounds were observed much earlier and at much lower concentrations in hepatocytes taken from mice and rats that had been pretreated with clofibric acid (p < or = 0.001, Sprague-Dawley rat and B6C3F1 mouse). DCA and TCA produced no evidence of cytotoxicity in hepatocytes from naive or clofibric acid-pretreated animals of either species at concentrations up to 5.0 mM. Increasing concentrations of MCA and GLY resulted in dose-related depletion of intracellular reduced glutathione (GSH) that closely paralleled the cytotoxic responses. Only GLY (0.25-5.0 mM) produced increased intracellular oxidized glutathione. Neither DCA nor TCA was found to alter cellular GSH status in hepatocytes isolated from either Sprague-Dawley rats or B6C3F1 mice. It was concluded from these in vitro observations that DCA and TCA are not highly cytotoxic to hepatocytes. Moreover, the rates of their conversion to MCA or GLY may be insufficient to induce cytotoxic effects in hepatocytes in vivo.