Renal involvement in systemic amyloidosis: an Italian collaborative study on survival and renal outcome.

BACKGROUND: Few data are available from large population-based studies on survival and renal outcome of patients with renal involvement and different types of systemic amyloidosis. METHODS: Two hundred and ninety of over 373 patients affected from systemic amyloidosis with renal involvement diagnosed in Italy between January 1995 and December 2000 were followed from diagnosis to death or until the last available clinical control. Eighty-three patients were excluded from analysis either because the amyloid type remained undetermined or they were lost at follow-up. Clinical and laboratory information was collected according to the different types of amyloidosis using a specific form which included renal function with 24 h proteinuria at diagnosis and at the end of follow-up, the type and the date of onset of dialysis and the kind of treatment they underwent. RESULTS: The median time of follow-up was 24 months in primary (AL) amyloidosis (range: 1-88 months), 16 months in AL with associated multiple myeloma (MM + AL: range 1-76 months), 30 months in reactive (AA) amyloidosis (range: 1-99 months) and 52 months in patients with familial forms (AF: range 14-82 months). Patients with AL showed a significantly shorter survival than AA. Despite no significant differences of renal outcome or survival on dialysis being observed between the two groups, a lower renal survival with a higher number of patients who progressed to end-stage renal disease (ESRD) was observed in patients with AA. Overall survival was markedly improved in patients with AL who underwent a specific therapy (conventional chemotherapy or autologous stem cell transplantation (ASCT)) even in the absence of a positive kidney response. Multivariate analysis showed cardiac involvement and specific therapy to significantly influence survival in AL whereas age, serum creatinine (sCr) and heart involvement significantly affected survival in AA. In both groups, sCr and heart involvement were the most relevant predictors for renal outcome, together with urinary protein excretion, in patients with AA. CONCLUSIONS: Our results show a worse survival in AL due to the higher prevalence of heart involvement in this group and emphasize that a specific therapy significantly prolongs survival and slows the progression of renal disease in patients with AL. We suggest that a late nephrological referral is likely the cause of the higher sCr found at presentation in patients with AA and probably accounts for the lower renal survival observed in the short term in these patients. At the time being, renal transplantation and ASCT are still rare therapeutic options for renal patients affected from systemic amyloidosis.

[Breathing sleep disturbances and occupational medicine: study of 20 clinical cases]. / Disturbi respiratori del sonno e medicina del lavoro: considerazioni su 20 casi clinici.

During 2004, in the Center for Sleep Disorders, a questionnaire including Epworth sleepiness scale (ES) was administered to 120 subjects; 20 male subjects of this group with elevated score (ES >14) were selected and submitted to polysomnography. Subjects, all in working age, were represented by 3 (15%) shift-workers, 9 (45%) drivers, 17 (85%) industrial workers (among those 5 building workers) and 3 (15%) employers. By polysomnography, moderatelsevere OSAHS was diagnosed in all subjects (40% moderate, 60% severe). CPAP (Continuous Positive Airway Pressure) therapy led to an improvement of clinical symptoms since the first month. Counselling of Occupational Medicine Physician with the Center for Sleep Disorders, was useful to direct the action of Competent Doctor, especially for jobs requiring high vigilance (drivers or shift-worker). The pass certificate for jobs with an high risk (alone, in high places, heavy means drivers) cannot avoid to evaluate this pathology, that is often associated to other related risk factors (obesity, hypertension, diabetes), because it compromises both the specific suitability and the protection of common health and safety.

Extrapolation of a PBPK model for dioxins across dosage regimen, gender, strain, and species.

A physiologically based pharmacodynamic (PBPK) model for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was developed based on pharmacokinetic data from acute oral exposures of TCDD to female Sprague-Dawley rats (Wang et al., 1997, Toxicol Appl. Pharmacol 147, 151-168). In the present study, the utility of this model to predict the disposition of TCDD in male and female Sprague-Dawley and female Wistar rats exposed to TCDD through different dosage regimens was examined. The ability of the model to predict the disposition of 2-iodo-3,7,8-trichlorodibenzo-p-dioxin (ITrCDD) in mice (Leung, et al., 1990, Toxicol. Appl. Pharmacol. 103, 399-410) was also examined. The ability of the model to predict across routes of exposure was assessed with intravenous injection data (5.6 microg/kg bw) (Li et al., 1995, Fundam. Appl. Toxicol. 27, 70-76) in female rats. Analysis across gender extrapolations used data for male Sprague-Dawley rats exposed intravenously to 9.25 microg TCDD/kg bw (Weber et al., 1993, Fundam. Appl. Toxicol. 21, 523-534). The analysis of across-dosage regimen and stains of rats extrapolations were assessed using data from rats exposed to TCDD through a loading/maintenance dosage regimen (Krowke et al., 1989, Arch. Toxicol. 63, 356-360). The physiological differences between gender, strain, and species were taken into account when fitting the PBPK model to these data sets. The results demonstrate that the PBPK model for TCDD developed for female Sprague-Dawley rats exposed by acute oral dosing accurately predicts the disposition of TCDD, for different gender and strain of rats across varying dosage regimens, as well as in a strain of mice. Minimal changes in fitted parameters were required to provide accurate predictions of these data sets. This study provides further confirmation of the potential use of physiological modeling in understanding pharmacokinetics and pharmacodynamics.

Dose-dependent localization of TCDD in isolated centrilobular and periportal hepatocytes.

Dose-response relationships for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest a differential sensitivity of liver cell types to the induction of cytochrome P450 gene expression, and that the induction of hepatic protein CYP1A2 causes sequestration of TCDD. In addition, immunolocalization of hepatic CYP1A1/1B1/1A2 proteins is not uniform after exposure to TCDD. The mechanism for the regio-specific induction of hepatic P450s by TCDD is unknown, but may involve the differential distribution of participants in the AhR-mediated pathway and/or regional P450 isozymes, as well as, non-uniform distribution/sequestration of TCDD. Therefore, this study examined the effects of TCDD in unfractionated, centrilobular and periportal hepatocytes isolated from female Sprague-Dawley rats acutely exposed (3 days) to a single oral dose of 0.01-10.0 microg [3H]TCDD/kg. A dose-dependent increase in concentration of TCDD was accompanied by a dose-dependent increase in CYP1A1, CYP1A2, and CYP1B1 mRNA expression and associated enzymes in all liver-cell populations. Centrilobular hepatocytes showed a 2.7- to 4.5-fold higher concentration of TCDD as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Centrilobular hepatocytes also exhibited an elevated MROD activity as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Furthermore, centrilobular hepatocytes showed an elevated concentration of induced CYP1A2 and CYP1B1 mRNA as compared to periportal hepatocytes within the 0.01- and 0.3-microg TCDD/kg-treatment groups. This is the first study to demonstrate that a dose-dependent difference in distribution of TCDD exists between centrilobular and periportal cells that might be related to regional differences in P450 induction.

Daily cycle of bHLH-PAS proteins, Ah receptor and Arnt, in multiple tissues of female Sprague-Dawley rats.

The aryl hydrocarbon receptor (AhR) shares a common PAS domain with a number of genes that exhibit a pronounced circadian rhythm. Therefore, this study examined the daily cycle of AhR and AhR nuclear translocator (Arnt) protein expression in multiple tissues of female Sprague-Dawley rats. Rats were euthanized at 4, 7, and 11 am and 4, 7, and 11 pm after which whole tissue homogenates were made from multiple tissues. Western blot analysis showed that the daily cycle of relative AhR protein expression exhibits a similar oscillation pattern in the liver, lungs, and thymus. The daily cycle of relative Arnt protein expression exhibits a similar oscillation pattern in the liver and lungs. The apparent daily cycle of AhR and Arnt protein expression in multiple tissues was not observed within the spleen. This preliminary report is the first study to suggest that the PAS proteins, AhR and Arnt, exhibit a daily oscillation pattern within multiple target tissues which may give insight into the tissue-specific toxic and biochemical responses mediated through this dimerization pair, as well as the physiological function of these proteins.

A pharmacodynamic analysis of TCDD-induced cytochrome P450 gene expression in multiple tissues: dose- and time-dependent effects.

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) to alter gene expression and the demonstration that the induction of CYP1A2 is responsible for hepatic TCDD sequestration suggest that both pharmacokinetic and pharmacodynamic events must be incorporated for a quantitative description of TCDD disposition. In this paper, a biologically based pharmacodynamic (BBPD) model for TCDD-induced biochemical responses in multiple tissues was developed. The parameters responsible for tissue response were estimated simultaneously with a refined physiologically based pharmacokinetic (PBPK) model developed by Wang et al. (1997a), by using the time-dependent effects of TCDD on induced CYP1A1/CYP1A2 gene expression in multiple target tissues (liver, lungs, kidneys, and skin) of female Sprague-Dawley rats treated with 10 microgram TCDD/kg for 30 min, 1, 3, 8, or 24 h, or 7, 14, or 35 days. This refined BBPD model developed based on the time-course of TCDD-induced CYP1A1/CYP1A2 protein expression, and associated enzymatic activities well described the dose-dependent effects of TCDD on cytochrome P450 protein expression and associated enzyme activities in the multiple tissues of female Sprague-Dawley rats at 3 days following a single exposure to TCDD (0.01-30.0 micromgram TCDD/kg). This is the first BBPD model to quantitatively describe the time- and dose-dependent effects of TCDD on induced CYP1A1/CYP1A2 protein expression and associated enzyme activities in multiple target tissues for TCDD-induced biochemical responses.

Female Sprague-Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus, and lung.

There is currently little information concerning the time-dependent relationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) protein concentration in vivo. Therefore, female Sprague-Dawley rats were given a single oral dose of TCDD (10 micrograms/kg), and the AHR and ARNT protein concentrations in liver, spleen, thymus, and lung determined by Western blotting. In liver, the concentration of AHR protein was significantly reduced 8 and 24 h postdosing as compared to time-matched controls. In spleen and lung, the concentration of AHR protein was reduced 3, 8, 24, and 168 h posttreatment compared to time-matched controls but returned to control levels by 336 h. In thymus, reductions in AHR protein concentration were observed 8, 24, 168, and 336 h postdosing as compared to time-matched controls. Significant reductions in the concentration of ARNT protein were not observed in any of the TCDD-exposed tissues. Functional studies in cell culture showed that exposure of a mouse hepatoma cell line (Hepa-1c1c7) and a rat smooth muscle cell line (A-7) to TCDD (1 nM) for 12 days resulted in a 50% reduction in TCDD-inducible reporter gene expression following subsequent challenge by an additional dose of TCDD (1 nM). Collectively, these results show that (i) TCDD-mediated depletion of AHR occurs in vivo, (ii) AHR protein does not rapidly recover to pretreatment levels even though the tissue concentration of TCDD has fallen, and (iii) reduction in AHR protein concentration correlates with reduction in TCDD-mediated reporter gene expression in mammalian culture cells.

Determination of parameters responsible for pharmacokinetic behavior of TCDD in female Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic member of a class of planar and halogenated chemicals. Improvements in exposure assessment of TCDD require scientific information on the distribution of TCDD in target tissues and cellular responses induced by TCDD. Since 1980, several physiologically based pharmacokinetic (PBPK) models for TCDD and related compounds have been reported. Some of these models incorporated the induction of a hepatic binding protein in response to interactions of TCDD, the Ah receptor, and DNA binding sites and described the TCDD disposition in a biological system for certain data sets. Due to the limitations of the available experimental data, different values for the same physical parameters of these models were obtained from the different studies. The inconsistencies of the parameter values limit the application of PBPK models to risk assessment. Therefore, further refinement of previous models is necessary. This paper develops an improved PBPK model to describe TCDD disposition in eight target tissues. The interaction of TCDD with the Ah receptor and with hepatic inducible CYP1A2 were also incorporated into the model. This model accurately described the time course distribution of TCDD following a single oral dose of 10 microg/kg, as well as the TCDD concentration on Day 3 after six different doses, 0.01, 0.1, 0.3, 1, 10, and 30 microg TCDD/kg, in target tissues. This study extends previous TCDD models by illustrating the validity and the limitation of the model and providing further confirmation of the potential PBPK model for us in optimal experimental design and extrapolation across doses and routes of exposure. In addition, this study demonstrated some critical issues in PBPK modeling.

Differential time-course and dose-response relationships of TCDD-induced CYP1B1, CYP1A1, and CYP1A2 proteins in rats.

This study examined the relationship between dose- and time-dependent hepatic localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and expression of CYP1B1, CYP1A1 and CYP1A2 proteins. A dose-dependent increase in hepatic TCDD in female Sprague-Dawley rats treated with 0.01-30.0 microg TCDD/kg was observed. TCDD induced CYP1A1 protein in rats treated with 0.3 microg TCDD/kg or higher. TCDD induced CYP1A2 and CYP1B1 proteins in rats treated with 1.0 microg TCDD/kg or higher. The in vivo ED50 (microg TCDD/kg) for TCDD-induced CYP1A1, CYP1A2 and CYP1B1 proteins were 0.22, 0.40 and 5.19, respectively. Hepatic accumulation of TCDD reached a maximum at 8 hours post dosing with a t1/2 of approximately 10 days. TCDD-induced CYP1A1/CYP1A2 protein expression was increased time-dependently, reaching a maximum at 3 days after dosing and remaining elevated for 35 days. In contrast, TCDD-induced CYP1B1 protein showed significant expression at 3 days after dosing and decreased to basal concentrations by 35 days. This study demonstrates that TCDD exhibits differential dose-response and time-course relationships on hepatic localization and cytochrome P-450 protein expression.

Subcellular localization of TCDD differs between the liver, lungs, and kidneys after acute and subchronic exposure: species/dose comparisons and possible mechanism.

Subcellular localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds has been examined only in the liver. The objective of this study was (1) to examine and compare the subcellular distribution of TCDD within hepatic and nonhepatic (lungs/kidneys) tissues of female Sprague-Dawley rats acutely exposed to TCDD, (2) to analyze species comparisons in the subcellular localization of TCDD in multiple tissues, (3) to investigate the effect of dose on subcellular distribution of TCDD, (4) to analyze the effect of subchronic exposure on the subcellular distribution of TCDD, and (5) to examine one possible mechanism for subcellular localization of TCDD. Female Sprague-Dawley rats and B6C3F1 mice received a single oral dose of 0.1, 1.0, or 10 microg [3H]TCDD/kg body weight and subcellular fractions of the liver, lungs, and kidneys were prepared by differential centrifugation 3 days after exposure. Analysis of the rat subcellular fractions revealed that TCDD was equally distributed between the hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9 (cytosol and microsomal) fractions at all doses tested. In contrast, TCDD was concentrated in the P9 of rat nonhepatic tissues at all doses studied. Differential centrifugation of the hepatic S9 showed that TCDD was localized within the hepatic P100 (microsomal) fraction at all doses tested. In contrast, TCDD localized in pulmonary and renal S100 (cytosolic) fractions at all doses. The subcellular distribution of TCDD in the liver and lungs of acutely exposed B6C3F1 mice was similar to that observed in the rats. Although TCDD was concentrated within the renal P9, the remainder of TCDD in the S9 was evenly distributed between the S100 and the P100 fractions of acutely exposed B6C3F1 mice. Subchronic exposure of B6C3F1 mice to 1.5 or 150 ng [3H]TCDD/kg/day revealed that increasing dose resulted in equal distribution of TCDD between the hepatic S9 and P9 versus concentration in the renal P9. In addition, a dose-dependent increase in accumulation of TCDD in the hepatic P100 was accompanied by a dose-dependent increase in TCDD localization in the renal S100 of mice subchronically exposed to TCDD. TCDD exposure in rats resulted in a dose-dependent increase in the induction of CYP1A1 protein and associated enzyme activity in hepatic, pulmonary, and renal microsomes. TCDD-induced CYP1A2 protein levels and associated enzymatic activity were only present in hepatic microsomes. This is the first report to suggest that subcellular distribution of TCDD differs between hepatic and nonhepatic tissues and demonstrate that the liver-specific microsomal localization of TCDD in female Sprague-Dawley rats also occurs in the liver of female B6C3F1 mice acutely or subchronically exposed to TCDD. In addition, these data are consistent with the hypothesis that the hepatic sequestration of TCDD is due to an interaction with CYP1A2. Furthermore, the lack of pulmonary/renal sequestration coupled with the lack of localization of TCDD in pulmonary/renal microsomes also supports the role of CYP1A2 as a hepatic microsomal binding protein involved in TCDD sequestration..

Characterization of the molecular and structural properties of the transformed and nuclear aryl hydrocarbon (Ah) receptor complexes by proteolytic digestion.

Ligand-dependent differences in the molecular properties of the transformed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were investigated using the proteolytic clipping band shift assay. AhR complexes were incubated with [32P]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crosslinked protein-DNA complexes were treated with trypsin or V8 protease and analyzed by electrophoresis. The results showed that for several different AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran and alpha-naphthoflavone, the pattern of degraded protein-DNA products were similar using transformed cytosolic or nuclear AhR complexes. In contrast, the proteolytic clipping band shift assay showed that there were significant differences in the pattern of degraded protein-DNA products using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and in vitro activities of these compounds in which TCDD is a potent AhR agonist whereas MCDF is a partial AhR agonist and antagonist.

Substituted flavones as aryl hydrocarbon (Ah) receptor agonists and antagonists.

The structure-dependent aryl hydrocarbon (Ah) receptor agonist and antagonist activities of the following substituted flavones were investigated: flavone, 4'-methoxy-, 4'-amino-, 4'-chloro-, 4'-bromo-, 4'-nitro-, 4'-chloro-3'-nitro-, 3'-amino-4'-hydroxy-, 3',4'-dichloro-, and 4'-iodoflavone. The halogenated flavones exhibited competitive Ah receptor binding affinities (IC50 = 0.79 to 2.28 nM) that were comparable to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (1.78 nM). The compounds also induced transformation of the rat cytosolic Ah receptor and induced CYP1A1 gene expression in MCF-7 human breast cancer cells. However, despite the high Ah receptor binding affinities for these responses, the halogenated flavones were > 1000 times less active than TCDD for the other responses. Moreover, for other substituted flavones, there was no correlation between Ah receptor binding affinities and their activities as Ah receptor agonists. For example, 4'-aminoflavone induced CYP1A1 mRNA levels in MCF-7 cells but exhibited relatively low Ah receptor binding affinity (IC50 = 362 nM) and did not induce transformation of the rat cytosolic Ah receptor. All of the substituted flavones inhibited TCDD-induced transformation of the Ah receptor, and 4'-iodoflavone, an Ah receptor agonist at high concentrations (1-50 microM), inhibited the transformation at concentrations as low as 0.05 and 0.5 microM. Subsequent interaction studies with TCDD and 4'-iodoflavone confirmed that the latter compound inhibits induction of CYP1A1 gene expression by TCDD in MCF-7 cells. The results obtained for the substituted flavones suggest that within this structural class of compounds, various substituent groups can affect markedly the activity of each individual congener as an Ah receptor agonist or antagonist. These substituent-dependent differences in activity may be related to ligand-induced conformational changes in the Ah receptor complex and/or support the proposed existence of more than one form of the Ah receptor.

Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells.

17 beta-Estradiol (E2) induces cathepsin D mRNA levels and intracellular levels of immunoreactive protein in MCF-7 human breast cancer cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression in this cell line; however, in cells cotreated with TCDD and E2, TCDD inhibited E2-induced cathepsin D mRNA levels, the rate of gene transcription, and levels of immunoreactive protein. The inhibitory responses were observed within 30 to 120 min after the cells were treated with TCDD. TCDD also inhibited E2-induced secreted alkaline phosphatase activity in aryl hydrocarbon (Ah)-responsive MCF-7 and wild-type mouse Hepa 1c1c7 cells cotransfected with the human estrogen receptor (hER) and the pBC12/S1/pac plasmid, which contains the 5' promoter region (-296/+57) of the cathepsin D gene and an alkaline phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 to -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-175/-181) xenobiotic responsive element (XRE); the role of this sequence in Ah responsiveness was investigated in gel electrophoretic mobility shift assays and with plasmid constructs containing a wild-type ER/Sp1 oligonucleotide or a mutant ER/Sp1-"XRE" oligonucleotide containing two C-->A mutations in the XRE sequence (antisense strand). In plasmid constructs which contained a chloramphenicol acetyltransferase reporter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chloramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-"XRE" plasmids. Electrophoretic mobility shift assays showed that the nuclear or transformed cytosolic Ah receptor complex blocked formation of the ER-Sp1 complex with the wild-type but not the ER/Sp1 mutant oligonucleotide. Moreover, incubation of the wild-type bromodeoxyuridine-substituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demonstrate that Ah receptor-mediated inhibition of E2-induced cathepsin D gene expression is due to disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.

Comparative properties of the nuclear aryl hydrocarbon (Ah) receptor complex from several human cell lines.

The aryl hydrocarbon (Ah) responsiveness of the T-47D, Hep G2, LS180, MCF-7, A431, C-4II and MDA-MB-231 human cancer cell lines was determined by the induction of CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity. With the exception of teh MDA-MB-231 breast cancer cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity in the remaining six cell lines and, based on their EC50 values, for ethoxyresorufin O-deethylase induction, their Ah responsiveness followed the order T-47D > C-4II > MCF-7 > LS180 > HEP G2 > A431. In contrast, all the cell lines expressed the nuclear Ah receptor complex (167.1-24.5 fmol/mg protein) which bound to a 32P-labeled consensus dioxin responsive element (DRE) in a gel mobility shift assay. The results of gel permeation chromatography a sucrose density gradient centrifugation studies showed that the calculated Mr values for the nuclear Ah receptor complex varied from 175 kDa (MDA-MB-231 cells) to 221 kDa and the apparent molecular weight of the nuclear Ah receptor complex cross-linked to a bromodeoxyuridine-substituted DRE was 200 kDa. The data show that the molecular properties and levels of the nuclear Ah receptor complex from seven different human cancer cell lines do not predict Ah responsiveness.

Induction of Cyp1a-1 and Cyp1a-2 gene expression by a reconstituted mixture of polynuclear aromatic hydrocarbons in B6C3F1 mice.

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs--indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs--acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); > or = 4-ring PAHs--fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The > or = 4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and > or = 4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the > or = 4-ring PAHs. The reconstituted PAH mixture and > or = 4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the > or = 4 ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.

Identification of 3'-methoxy-4'-nitroflavone as a pure aryl hydrocarbon (Ah) receptor antagonist and evidence for more than one form of the nuclear Ah receptor in MCF-7 human breast cancer cells.

The competitive binding of 3'-methoxy-4'-nitro, 4'-amino-3'-methoxy, 4'-methoxy-3'-nitro, and 3'-amino-4'-methoxyflavone (compounds 1 to 4, respectively) to the rat cytosolic aryl hydrocarbon (Ah) receptor gave IC50 values of 2.27, 86.1, 872, and 19.4 nM. Flavones 3 and 4 were characterized as Ah receptor agonists in MCF-7 human breast cancer cells and induced CYP1A1 gene expression, whereas the 3-methoxy-substituted flavones (1 and 2) were inactive. All four compounds inhibited induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells; moreover, in vitro studies with TCDD-induced rat liver microsomes showed that flavones 1 to 4 inhibited EROD activity in the presence or absence of NADPH. In MCF-7 cells cotreated with flavones 1 or 2 (0.01 to 10 microM) plus TCDD or [3H]TCDD, there was a concentration-dependent inhibition of TCDD-induced CYP1A1 mRNA levels and formation of radiolabeled nuclear Ah receptor complex. Velocity sedimentation analysis of nuclear extracts from MCF-7 cells treated with [3H]TCDD plus 1 or 10 microM concentrations of flavones 1 and 2 showed that an early eluting specifically bound nuclear Ah receptor complex was present. However, under these same conditions the flavones inhibited TCDD-induced CYP1A1 gene expression. The apparent molecular mass of this nuclear complex was 190 kDa as determined by cross-linking to a 32P-labeled bromodeoxyuridine-substituted consensus dioxin-responsive element. Similar cross-linking results were obtained using the nuclear extract from MCF-7 cells treated with [3H]TCDD alone. The results of this study suggest that there are at least two forms of the nuclear Ah receptor complex in MCF-7 cells; the major transcriptionally active form binds [3H]TCDD and flavones 1 or 2 inhibit nuclear uptake of this receptor complex. The other form of the nuclear Ah receptor complex appears to be transcriptionally inactive and ligand binding with [3H]TCDD is not competitively inhibited by flavones 1 and 2.

Aryl hydrocarbon (Ah) receptor-independent induction of Cyp1a2 gene expression by acenaphthylene and related compounds in B6C3F1 mice.

Treatment of B6C3F1 mice with acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene and dibenzofuran resulted in induction of hepatic microsomal methoxyresorufin O-deethylase (MROD) activity. Acenaphthylene was the most potent inducer of MROD, a Cyp1a2-dependent activity, and was utilized as a prototypical inducer for this group of tricyclic hydrocarbons. Acenaphthylene (300 mg/kg) caused a > 80-fold induction of hepatic microsomal MROD activity; no induction was observed in kidney or lung. Analysis of induced hepatic microsomes with antibodies to Cyp1a1 and Cyp1a2 showed that acenaphthylene induced immunoreactive Cyp1a2 but not Cyp1a1 proteins and subsequent mRNA analysis confirmed with a cDNA probe for Cyp1a1 and Cyp1a2 that acenaphthylene induced Cyp1a2 but not Cyp1a1 mRNA. Results from nuclear run-on experiments using hepatic nuclei showed that acenaphthylene caused an approximately 4-fold increase in the rate of Cyp1a2 gene transcription in B6C3F1 mice. Results of competitive binding studies indicated that the tricyclic hydrocarbons did not competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin or [3H]benzo[a]pyrene from the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor or 4S carcinogen binding protein respectively. The data indicate that acenaphthylene and related tricyclic hydrocarbons induce Cyp1a2 gene expression in B6C3F1 mice via an Ah receptor-independent pathway. Thus, tricyclic hydrocarbons induce Cyp1a2 without the co-induction of Cyp1a1 and therefore these relatively non-toxic compounds can be used to further probe the role of Cyp1a2 in the metabolism and metabolic activation of diverse chemical carcinogens.

2-Phenylphenanthridinone and related compounds: aryl hydrocarbon receptor agonists and suicide inactivators of P4501A1.

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 microM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a > 80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.

Thrombotic thrombocytopenic purpura: a rare late complication of allogeneic bone marrow transplantation.

Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) usually occurs in a setting of systemic infection or graft-versus-host reaction during the first weeks following transplant. We report a case of fatal TTP that developed eight months after allogeneic bone marrow transplantation (BMT) without any evident association with other transplantation-related complications. Conditioning chemotherapy could have induced the disorder by causing damage to the vascular endothelium. The removal of immunosuppression, including cessation of cyclosporin A (CyA), may have precipitated the disease.