Henry d'A. Heck

Formaldehyde (CH2O) Concentrations in the Blood of Humans and Fischer-344 Rats Exposed to CH2O Under Controlled Conditions

The effect of exposure to formaldehyde (CH2O) on the CH2O concentration of the blood was determined. Eight male F-344 rats were exposed to 14.4 +/- 2.4 ppm of CH2O for 2 hours and the blood was collected immediately after exposure. Formaldehyde concentrations in the blood were determined by gas chromatography/mass spectrometry. The blood of eight rats unexposed to CH2O was collected and analyzed in the same manner. Measured CH2O concentrations (micrograms/g of blood) were: controls, 2.24 +/- 0.07; exposed, 2.25 +/- 0.07 (mean +/- S.E.). Formaldehyde concentrations in human blood were determined by analyzing samples of venous blood collected before and after exposure of six human volunteers (4 M, 2 F) to 1.9 +/- 0.1 ppm of CH2O for 40 min. Average CH2O concentrations (micrograms/g of blood) were: before exposure, 2.61 +/- 0.14; after exposure, 2.77 +/- 0.28. In neither experiment was there a statistically significant effect of exposure on the average CH2O concentration of the blood. However, human subjects differed significantly with respect to their blood CH2O concentrations, and significant differences (either an increase or a decrease) were found between the CH2O concentrations of the blood taken before and after exposure from some of the subjects, suggesting that blood CH2O concentrations may vary with time.

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

DNA-protein cross-links were formed in the respiratory tract of rhesus monkeys exposed to [14C]formaldehyde (0.7, 2, or 6 ppm; 6 hr). Concentrations of cross-links (pmol/mg DNA) were highest in the mucosa of the middle turbinates; lower concentrations were produced in the anterior lateral wall/septum and nasopharynx. Very low concentrations were found in the larynx/trachea/carina and in the proximal portions of the major bronchi of some monkeys exposed to 6 ppm but not to 0.7 ppm. No cross-links were detected in the maxillary sinuses or lung parenchyma. The pharmacokinetics of cross-link formation in the nose were interpreted using a model in which the rate of formation is proportional to the tissue concentration of formaldehyde. The model includes both saturable and nonsaturable elimination pathways and describes regional differences in DNA binding as having an anatomical rather than a biochemical basis. Using this model, the concentration of cross-links formed in corresponding tissues of different species can be predicted by scaling the pharmacokinetic parameter that depends on minute volume (V) and quantity of nasal mucosal DNA (MDNA). The concentration-response curve for the average rate of cross-link formation in the turbinates, lateral wall, and septum of rhesus monkeys was predicted from that of F-344 rats exposed under similar conditions. There was significant overlap between predicted and fitted curves, implying that V and MDNA are major determinants of the rate of cross-link formation in the nasal mucosa of different species. Concentrations of cross-links that may be produced in the nasal mucosa of adult men were predicted based on experimental data in rats and monkeys. The results suggest that formaldehyde would generate lower concentrations of cross-links in the nasal mucosa of humans than of monkeys, and much lower concentrations in humans than in rats. The rate of formation of DNA-protein cross-links can be regarded as a surrogate for the delivered concentration of formaldehyde. Use of this surrogate should decrease the uncertainty of human cancer risk estimates derived by interspecies extrapolation by providing a more realistic measure of the delivered concentration at critical target sites.

Differentiation between metabolic incorporation and covalent binding in the labeling of macromolecules in the rat nasal mucosa and bone marrow by inhaled [14C]- and [3H]formaldehyde

The mechanisms of labeling of macromolecules (DNA, RNA, and protein) in the respiratory and olfactory mucosa, and in the bone marrow (femur) of male Fischer-344 rats exposed to [14C]- and [3H]formaldehyde [( 14C]- and [3H]CH2O) were investigated. Animals were exposed for 6 hr to atmospheres containing [14C]- and [3H]CH2O at concentrations of 0.3, 2, 6, 10, or 15 ppm, 1 day following a single pre-exposure to the same concentration of unlabeled CH2O. The major route of nucleic acid labeling at all concentrations and in all tissues was metabolic incorporation; protein labeling in the respiratory mucosa was mainly due to covalent binding at the higher CH2O concentrations. Incorporation of [14C]CH2O into DNA in the respiratory mucosa was maximal at 6 ppm but decreased at higher concentrations, whereas labeling of DNA in the olfactory mucosa and bone marrow increased monotonically with concentration. Evidence for covalent binding of CH2O to respiratory mucosal DNA was obtained at CH2O concentrations equal to or greater than 2 ppm. The concentration of CH2O covalently bound to DNA at 6 ppm was 10.5-fold higher than at 2 ppm, indicating significant nonlinearity of DNA binding with respect to the inhaled formaldehyde concentration under these conditions. Covalent binding to proteins increased in an essentially linear manner with increases in the airborne concentration. No evidence was obtained for the formation of covalent adducts with macromolecules in the olfactory mucosa or bone marrow. The nonlinear increase in covalent binding to respiratory mucosal DNA with increasing CH2O concentrations may be explained either by a decrease in the efficiency of defense mechanisms or by an increase in the availability of reaction sites on the DNA resulting from increased cell turnover.

Oxidation of formaldehyde and acetaldehyde by NAD+-dependent dehydrogenases in rat nasal mucosal homogenates

Homogenates of respiratory and olfactory tissue from the rat nasal cavity were examined for their capacity to catalyze the NAD+-dependent oxidation of formaldehyde (in the presence and absence of glutathione) and of acetaldehyde. Both aldehydes were oxidized efficiently by nasal mucosal homogenates, and formaldehyde dehydrogenase (FDH) and aldehyde dehydrogenase (AldDH) were tentatively identified in both tissue samples. At least two isozymes of AldDH, differing with respect to their apparent Km and Vmax values with acetaldehyde as substrate, were found in the nasal mucosa, one of which may catalyze the oxidation of both formaldehyde and acetaldehyde. The specific activity of FDH in the olfactory mucosa was twice that in the respiratory mucosa, whereas the specific activity of the higher Km isozyme of AldDH was five to eight times greater in respiratory than in olfactory tissue. The specific activity of the lower Km isozyme of AldDH was similar in respiratory and olfactory homogenates. Repeated exposures of rats to formaldehyde (15 ppm, 6 hr/day, 10 days) or to acetaldehyde (1500 ppm, 6hr/day, 5 days) did not substantially affect the specific activities of FDH and AldDH in nasal mucosal homogenates. Glutathione is a cofactor for FDH; the concentration of nonprotein sulfhydryls in respiratory mucosal homogenates was approximately 2.8 mumoles/g and was not changed significantly by repeated exposures to formaldehyde (15 ppm, 6hr/day, 9 days). These data indicate that the rat nasal mucosa, which is the major target site for both aldehydes in inhalation toxicity studies, can metabolize both formaldehyde and acetaldehyde, and that the specific activities of formaldehyde and aldehyde dehydrogenase in homogenates of the nasal mucosa are essentially unchanged following repeated exposures to toxic concentrations of either compound.

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

Inhalation of 3HCHO and H14CHO (6 ppm, 6 hr) resulted in the formation of DNA-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 enzymatically hydrolyzed to deoxyribonucleosides in Tris buffer and analyzed by HPLC with liquid scintillation counting (LSC). HCHO was bound exclusively to the IF DNA, indicating that the HCHO was bound as DNA-protein crosslinks. Hydrolysis of the DNA quantitatively released the HCHO; no evidence was obtained for the formation of hydroxymethyl adducts. An adduct detected previously following incubation of mammalian cells with HCHO, N6-hydroxymethyldeoxyadenosine (hm6dA) [Beland, F.A., Fullerton, N.F., and Heflich, R.H. (1984) J. Chromatogr. 308, 121-131], was shown to be produced by reaction of HCHO with deoxyadenosine (dA) in bis-Tris buffer under conditions similar to those used for hydrolysis of the DNA. This reaction does not occur in Tris buffer. Evidence was obtained that most or all of the hm6dA observed can be explained by this reaction. Based on these results, an improved method to determine the amount of H14CHO bound to DNA was developed: the DNA is hydrolyzed in Tris buffer and analyzed by HPLC, and the released H14CHO is derivatized with dimedone and quantitated by LSC. Rats were exposed to a wide range of H14CHO concentrations (0.3, 0.7, 2, 6, or 10 ppm; 6 hr). DNA-protein crosslinking occurred at all concentrations. The formation of crosslinks was interpreted in terms of a nonlinear pharmacokinetic model incorporating oxidation of inhaled HCHO as a defense mechanism. The slope of the fitted concentration-response curve at 10 ppm is 7.3-fold greater than at 0.3 ppm, and the detoxication pathway is half-saturated at an airborne concentration of 2.6 ppm.

The induction of bladder stones by terephthalic acid, dimethyl terephthalate, and melamine (2,4,6-triamino-s-triazine) and its relevance to risk assessment

Terephthalic acid (TPA), dimethyl terephthalate (DMT), and melamine (MA) induced calculi and transitional cell hyperplasia in urinary bladders of rats. A high incidence of calculi was induced in weanling rats, but the incidence was much lower in adult rats ingesting the same dietary concentration of the chemical. The dose-response curves for the induction of urolithiasis in weanling rats were extremely steep, consistent with the fact that the formation calculi can occur in urine that is supersaturated, but not in urine that is undersaturated with respect to the stone components. In the cases of TPA and DMT, stones were composed primarily of calcium terephthalate (CaTPA). By determining the solubility of CaTPA, the concentration of TPA that would be required to achieve urinary saturation was calculated, and a conservative estimate of the amount of TPA or DMT that would have to be absorbed in order to induce calculi was derived. TPA and MA induced bladder tumors in rats in chronic feeding studies. However, it is likely that these tumors were secondary to the development of calculi. TPA and MA are apparently nongenotoxic, and they do not appear to be metabolized. Increased cell replication in the urothelium of the bladder caused by chronic physical injury was probably a major factor in the mechanism of induction of bladder tumors by bladder stones. Bladder neoplasms occurred primarily in the high dose groups, and they were usually, although not invariably, associated with stones. The possibility that stones were passed or were lost during processing of tissues for histopathologic examination could explain the absence of calculi from some of the neoplastic bladders. The formation of bladder calculi is an example of a threshold effect. Although there is strong evidence linking bladder stones with the induction of tumors, the existence of thresholds in chemical carcinogenesis continues to be controversial. A decision by the U.S. Environmental Protection Agency concerning the levels of MA allowed to occur in the food chain indicates that data regarding thresholds, even in the case of urolithiasis, are not being utilized in the risk assessment process.

Depletion of nasal mucosal glutathione by acrolein and enhancement of formaldehyde-induced DNA-protein cross-linking by simultaneous exposure to acrolein.

Incubation of homogenates of rat nasal mucosa with acrolein resulted in the apparent formation of DNA-protein cross-links. However, inhalation exposure of male Fischer-344 rats to acrolein (2.0 ppm, 6 h) did not cause detectable DNA-protein cross-linking in the nasal respiratory mucosa. Simultaneous exposure of rats to both acrolein (2.0 ppm) and formaldehyde (6.0 ppm) for 6 h resulted in a significantly higher yield of DNA-protein cross-links than was obtained following exposure to formaldehyde (6.0 ppm) alone. Acrolein exposure at concentrations of 0.1, 0.5, 1.0, or 2.5 ppm resulted in a concentration-dependent depletion of nonprotein sulfhydryl groups in the nasal respiratory mucosa. A plausible explanation for the enhancement of DNA-protein cross-links by simultaneous exposure to formaldehyde and acrolein may be that depletion of glutathione by acrolein inhibited the oxidative metabolism of formaldehyde, leading to an increase of formaldehyde-induced DNA-protein cross-links.

Effects of short-term benzene administration on bone marrow cell cycle kinetics in the rat.

Abstract Hematology, cell cycle phase, and [ 3 H]thymidine ([ 3 H]TdR) uptake and retention in bone marrow were studied in male Fischer-344 rats exposed to benzene by repeated subcutaneous injection (0.5 or 1.0 ml benzene/kg/day). Peripheral lymphocytes and differentiating bone marrow precursor cells were found to be the most sensitive cell populations following repeated benzene administration. Benzene exposure resulted in an increase in the relative number of bone marrow precursor cells in G 2 or M phase of the cell cycle as determined by laser-based flow cytofluorometry. Benzene treatment resulted in an increase in cell proliferative activity as determined by both cytofluorometry and [ 3 H]TdR uptake. Although the uptake of [ 3 H]TdR into DNA was initially higher in animals repeatedly exposed to benzene than in controls, paralleling the increase in cell proliferative index, the specific activity of DNA rapidly decreased, suggesting a defect in maturation among affected precusor cells. A general inhibition of DNA synthesis in bone marrow was not observed. It would appear that benzene-induced cytotoxicity in bone marrow is a function of both cell differentiation and cell cycle phase.

Effects of formaldehyde exposure on the extractability of DNA from proteins in the rat nasal mucosa

Reaction of an homogenate of the rat nasal mucosa with formaldehyde (CH2O) followed by solubilization and extraction in a strongly denaturing aqueous-immiscible organic solvent mixture decreased the quantity of nucleic acids that remained in aqueous solution in comparison with a control homogenate untreated with CH2O. The absent DNA and RNA were located in the interface between the aqueous and organic phases, from which they could be recovered only after enzymatic proteolysis. It is concluded that interfacial nucleic acids were cross-linked to proteins by CH2O. The concentration of cross-links was estimated with 14CH2O; under conditions that rendered 48% of the DNA nonextractable from proteins, there was less than one cross-link per 28,000 nucleotide residues. Exposure of rats to airborne CH2O at concentrations of 0, 2, 6, 15, and 30 ppm (6 hr/day for 2 days) resulted in a statistically significant increase in the percentage of the total DNA from the respiratory mucosa that was located in the interface at concentrations of 6 ppm and higher. However, the percentage of DNA from the olfactory mucosa located in the interface was not increased by CH2O exposure. Analysis of nasal mucosal DNA by ultracentrifugation in CsCl density gradients provided no evidence of a change in the buoyant density of the DNA caused by reaction with CH2O. The results indicate that CH2O may induce DNA-protein cross-links in the respiratory but not in the olfactory mucosa at concentrations equal to or greater than 6 ppm.

HISTOCHEMICAL LOCALIZATION OF FORMALDEHYDE DEHYDROGENASE IN THE RAT

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowmans and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.