Covalent Binding of Inhaled Formaldehyde to DNA in the Nasal Mucosa of Fischer 344 Rats: Analysis of Formaldehyde and DNA by High-Performance Liquid Chromatography and Provisional Pharmacokinetic Interpretation

Covalent Binding of Inhaled Formaldehyde to DNA in the NasalMucosa of Fischer 344 Rats: Analysis of Formaldehyde and DNAby High-Performance Liquid Chromatography and Provisional PharmacokineticInterpretation. CASANOVA, M., DEYO, D. F., AND HECK, H. D'A.(1989). Fundam Appl. Toxicol. 12, 397–417. Inhalationof ³HCHO and H¹⁴CHO(6 ppm, 6 hr) resulted in the formation ofDNA-protein crosslinks in the rat nasal respiratory mucosa.The DNA was extracted and was fractionated into aqueous (AQ)and interfacial (IF) portions. AQ DNA and IF DNA were enzymaticallyhydrolyzed to deoxyribonucleosides in Tris buffer and analyzedby HPLC with liquid scintillation counting (LSC). HCHO was boundexclusively to the IF DNA, indicating that the HCHO was boundas DNA-protein crosslinks. Hydrolysis of the DNA quantitativelyreleased the HCHO; no evidence was obtained for the formationof hydroxymethyl adducts. An adduct detected previously followingincubation of mammalian cells with HCHO, N⁶-hydroxymethyldeoxyadenosine(hm6dA)[Beland F. A., Fullerton, N. F., and Heflich, R. H. (1984) J.Chromartogr. 308 121–131], was shown to be produced byreaction of HCHO with deoxyadenosine (dA) in bis-Tris bufferunder conditions similar to those used for hydrolysis of theDNA. This reaction does not occur in Tris buffer. Evidence wasobtained that most or all of the hm6dA observed can be explainedby this reaction. Based on these results, an improved methodto determine the amount of H¹⁴CHO bound to DNA was developed:the DNA is hydrolyzed in Tris buffer and analyzed by HPLC, andthe released H¹⁴CHO is derivatized with dimedone and quantitatedby LSC. Rats were exposed to a wide range of H¹⁴CHO concentrations(0.3, 0.7, 2, 6, or 10 ppm; 6 hr). DNA-protein crosslinkingoccurred at all concentrations. The formation of crosslinkswas interpreted in terms of a nonlinear pharmacokinetic modelincorporating oxidation of inhaled HCHO as a defense mechanism.The slope of the fitted concentration-response curve at 10 ppmis 7.3-fold greater than at 0.3 ppm, and the fitted detoxicationpathway is half-saturated at an airborne concentration of 2.6ppm.     

Covalent Binding of Inhaled Formaldehyde to DNA in the Respiratory Tract of Rhesus Monkeys: Pharmacokinetics, Rat-to-Monkey Interspecies Scaling, and Extrapolation to Man

Covalent Bindingoflnhaled Formaldehyde to DNA in the RespiratoryTractofRhesus Monkeys: Pharmacokinetics, Rat-to-Monkey InterspeciesScaling, and Extrapolation to Man. CASANOVA, M., MORGAN, K.T., STEINHAGEN, W. H., EVERITT, J. I., POPP, J. A., AND HECK,H. D'A. (1991). Fundam. Appl Toxicol 17, 409–428. DNA-proteincross-links were formed in the respiratory tract of rhesus monkeysexposed to [¹⁴C]formaldehyde (0.7, 2, or 6 ppm; 6 hr). Concentrationsof cross-links (pmol/mg DNA) were highest in the mucosa of themiddle turbmates; lower concentrations were produced in theanterior lateral wall/septum and nasopharynx. Very low concentrationswere found in the larynx/trachea/carina and in the proximalportions of the major bronchi of some monkeys exposed to 6 ppmbut not to 0.7 ppm. No cross-links were detected in the maxillarysinuses or lung parenchyma. The pharrnacokinetics of cross-linkformation in the nose were interpreted using a model in whichthe rate of formation is proportional to the tissue concentrationof formaldehyde. The model includes both saturable and nonsaturableelimination pathways and describes regional differences in DNAbinding as having an anatomical rather than a biochemical basis.Using this model, the concentration of cross-links formed incorrespondmg tissues of different spacies can be predicted byscaling the pharmacokinetic parameter that depends on minutevolume () and quantity of nasal mucosal DNA (M_DNA). The concentration-response curve for the average rateof cross-link formation in the turbinates lateral wall, andseptum of rhesus monkeys was predicted from that of F-344 ratsexposed under similar conditions. There was significant overlapbetween predicted and fitted curves, implying that V and M_DNAare major determinants of the rate of cross-link formation inthe nasal mucosa of different species. Concentrations of cross-linksthat may be produced in the nasal mucosa of adult men were predictedbased on experimental data in rats and monkeys. The resultssuggest that formaldehyde would generate lower concentrationsof cross-links in the nasal mucosa of humans than of monkeys,and much lower concentrations in humans than in rats. The rateof formation of DNA-protein cross-links can be regarded as asurrogate for the delivered concentration of formaldehyde. Useof this surrogate should decrease the uncertainty of human cancerrisk estimates derived by interspecies extrapolation by providinga more realistic measure of the delivered concenmtion at criticaltarget sites.     

Decreased Extractability of DNA from Proteins in the Rat Nasal Mucosa after Acetaldehyde Exposure

Decreased Extractability of DNA from Proteins in the Rat NasalMucosa after Acetaldehyde Exposure. LAM, C.-W., CASANOVA, M.,AND HECK, H. D'A. (1986). Fundam. Appl. Toxicol. 6, 541–550.Acetaldehyde and formaldehyde have been found to induce nasalcancer in two species of rodents. To understand the mechanismof carcinogenesis by acetaldehyde, studies were carried Outto determine whether acetaldehyde can react with DNA in targettissues of the rat nasal cavity. When fresh homogenates of thenasal respiratory mucosa were incubated with acetaldehyde (distilledunder N at concentrations of 10, 100, or 500 mM, followed bysolubilization and extraction with a strongly denaturing aqueous-immiscibleorganic solvent mixture, a decrease was observed in the amountof DNA partitioned into the aqueous phase at the two higheracetaldehyde con centrations. The absent DNA was recovered fromthe interfacial layer by proteolytic digestion. Similarly, incubationof calf thymus nucleohistones with acetaldehyde (100, 300, or1000 mM) or with formaldehyde (10, 30, or 100 mM) followed byprecipitation of the DNA with H₂SO₄ and analysis of the supematantsby sodium dodecyl sulfate-polyacrylamide gel electrophoresisresulted in concentration-dependent decreases in the quantitiesof histone proteins released from the DNA. These results indicatethat acetaldehyde as well as formaldehyde can form DNA-proteincrosslinks in vitro. A single 6-hr exposure of male Fischer-344rats to acetaldehyde (100, 300, 1000, or 3000 ppm) resultedin a significant increase relative to air-exposed controls inthe percent interfacial DNA from the nasal respiratory mucosaat concentrations equal to or greater than 1000 ppm. No increasein the interfacial DNA from the olfactory mucosa was detectedafter a single 6-hr exposure (1000 or 3000 ppm), but a significantincrease was found in rats exposed repeatedly (6 hr/day for5 days) to acetaldehyde (1000 ppm). Thus, evidence has beenobtained for the formation of DNA-protein crosslinks by acetaldehydein target tissues of the rat nasal cavity at concentrationssimilar to those that induced nasal cancer.     

CHLORACNE IN THE 1990s

Background. Chloracne is a disease associated with toxicity of halogenated compounds used in some industrial processes. A patient affected by chloracne led us to study a total of nine cases from a single factory. Methods. We studied the clinical features of nine patients exposed chronically to chlorobenzenes. On all of them blood samples were drawn and biopsies of affected skin and liver were taken. Their work environment was visited and studied. Results. All nine patients were men and had polymorphic skin lesions, characterized mainly by comedones and cysts. They had chronic conjunctivitis and seven had cysts in the Meibomian glands. All of them had polyneuropathy and liver damage and seven had hypertriglyceridemia. Compounds known to cause chloracne were found in exceedingly high concentrations in the water used in the workplace. Conclusions. Every patient exposed to halogenated compounds with the cutaneous manifestations of chloracne should be carefully investigated for systemic complications (such as ophthalmic, neuropathic, hepatic, and lipoprotein abnormalities).     

Low-dose interferon alpha combined with zidovudine in patients with AIDS-associated Kaposi's sarcoma

Abstract. Forty patients with AIDS-associated Kaposi's sarcoma (KS) treated with the combination of interferon alpha-2b (IFN-α) 10–20 MU day^?1 and zidovudine (ZDV), 500–800 mg day^?1, were evaluated for safety and efficacy. Eighteen patients (45%) had an overall response (CR + PR) at 3 months and a response persisting for a median of 14 (3–27) months. Patients with a CD4 count of less than 300 mm^?3, prior to opportunistic infections or constitutional symptoms, were less likely to respond. However, between 28.5% and 36% of patients with a low CD4 count did respond to combined therapy. This is higher than would be predicted from single agent IFN-α therapy. Twelve of 28 patients (42.8%) receiving 10 MU day^?1 of IFN-α (low dose) had an overall response. In addition, patients tolerated this dose of IFN-α better, presenting fewer flu-like symptoms and displayed a trend toward less anaemia, p24 antigen decreased in six out of nine evaluable cases, four of whom were treated with low-dose IFN-α. Low-dose IFN-α plus ZDV seems to be a useful and well-tolerated therapy for KS with antitumoral and antiviral activity. Patients without ‘bad prognostic markers’ are most likely to show improvement.