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Survival, DNA synthesis and ribosomal RNA transcription in monkey kidney cells treated by formaldehyde.

Separate cultures of CV-1 cells were exposed for 15 min to 1-16 mM formaldehyde (FA) at various time intervals before labeling with [3H]uridine. The labeled RNA extracted from whole cells was analyzed by polyacrylamide gel electrophoresis. Results indicated that FA produced transcription-terminating lesions in DNA which depressed RNA synthesis, gave rise to shortened RNA chains and modified the expression of transcription-linked genes. These lesions were efficiently repaired since a recovery of RNA transcription, with disappearance of the initially observed alterations, rapidly occurred during post-treatment incubation of cells. Cycloheximide (5 micrograms/ml) strongly inhibited this recovery, whereas fluorodeoxyuridine (10(-5) M) was without effect. The rate of semi-conservative DNA synthesis, measured autoradiographically by thymidine incorporation, as well as the number of cells performing DNA replication, fell to zero after 15-min exposure to FA concentrations greater than 2 mM. Both parameters recovered subnormal levels during a 24-h incubation after treatment with FA concentration up to 8 mM, in agreement with the high survival observed. Unscheduled DNA synthesis was not detectable during the restoration of DNA and RNA synthesis.

Animals↗

Formaldehyde mutagenesis in Drosophila. Molecular analysis of ADH-negative mutants.

The Adh gene from 4 formaldehyde-generated ADH-negative mutants of Drosophila melanogaster has been cloned and sequenced. All 4 mutants bear small deletions within the gene, ranging in size from 6 to 34 base pairs. 2 of the deletions lie within a 65-base pair intervening sequence and are accompanied by other aberrations. The other two are within the protein coding region of the gene. Some of these aberrations may be explained by a slipped mispairing mechanism.

Alcohol Dehydrogenase↗

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing.

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: approximately 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24 degrees C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38 degrees C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38 degrees C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.

Animals↗

Formaldehyde mutagenesis and formation of DNA-protein crosslinks in human lymphoblasts in vitro.

Human lymphoblasts were exposed in vitro to various concentrations of formaldehyde (HCHO) in single and multiple treatment regimens to determine relative mutagenic efficiency. Single treatments of HCHO (0-150 microM X 2 h) resulted in a nonlinear increase in induced mutant fraction at the thymidine kinase locus with increasing slope at concentrations above 125 microM. Only HCHO exposures of 125 microM X 2 h or greater produced significant effects on the growth rate of the lymphoblasts. Cultures were also exposed to either three treatments of 50 microM X 2 h, five treatments of 30 microM X 2 h, or ten treatments of 15 microM X 2 h; multiple treatments were administered on different days. These multiple treatments resulted in increases in mutant fraction, although their combined effect was less than a single treatment of equivalent concentration X time (150 microM X 2 h). Exposure of lymphoblasts to four treatments of 150 microM X 2 h HCHO failed to induce mutations at the ouabain resistance locus. Cultures of lymphoblasts receiving a single treatment of HCHO (0-600 microM X 2 h) were analyzed by the alkaline elution technique to detect the presence of DNA-protein crosslinks. HCHO treatment resulted in a significant nonlinear increase in DNA-protein crosslinks at concentrations greater than 50 microM X 2 h, which correlated with the onset of significant toxicity in this cell line. Holding the culture for 24 h resulted in complete removal of the crosslinks. These data indicate that both the induction of mutations and the formation of DNA-protein crosslinks by HCHO are nonlinear functions in human lymphoblasts and occur at overlapping concentration ranges.

Cells, Cultured↗

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

Animals↗

Tissue reactions to two root canal sealers containing formaldehyde.

Tissue reaction to root fillings using either Endomethasone or N2 was investigated in the premolar teeth of dogs. Periapical inflammation was frequently observed, but of greater significance was the universal evidence of ankylosis and the accompanying resorption of teeth filled with N2. One tooth filled with Endomethasone also exhibited this condition. The results of this study do not support the use of these root canal sealers that contain formaldehyde.

Administration, Topical↗

Nasal cavity deposition, histopathology, and cell proliferation after single or repeated formaldehyde exposures in B6C3F1 mice and F-344 rats.

Inhalation exposure for 2 years to 14.3 ppm formaldehyde (HCHO) induced a 50% incidence of squamous cell carcinoma in the nasal cavity of F-344 rats but only a 3.3% incidence in B6C3F1 mice. Since the response was concentration and species dependent, species differences in nasal cavity "dose" were examined as a possible mechanism for the differences in tumor incidence. Naive (nonpretreated) and HCHO-pretreated (6 or 15 ppm, 6 hr/day, 4 days) mice and rats were exposed to HCHO for 6 hr during which respiratory rate and tidal volume were recorded to calculate the theoretical deposition (micrograms/min/cm2) of HCHO on the nasal epithelium. Species differences in delivered "dose" were further assessed by comparative autoradiography, histopathology, and cell turnover studies. Because mice were better able to reduce minute ventilation upon repeated exposures, they had less HCHO available for deposition than rats, resulting in less tissue damage and a lower rate of cell turnover in the nasal epithelium. The correlation between calculated "dose" and observed nasal toxicity, including nasal tumor incidence, demonstrates that by normalizing the dosimetry to nasal surface area, species differences in nasal toxicity may be better understood.

Animals↗

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.

Animals↗

Sensory irritation tolerance and cross-tolerance in F-344 rats exposed to chlorine or formaldehyde gas.

Inhalation of chlorine (Cl2) or formaldehyde (HCHO) stimulates the trigeminal nerve endings in the nasal mucosa and results in respiratory rate depression in a concentration-dependent manner. To determine tolerance and cross-tolerance, the concentration-response curves of respiratory depression were compared between naive rats and rats pre-exposed to Cl2 or HCHO. Chlorine tolerance development was time and concentration dependent, being significant following a 1-day (6 hr/day), 10 ppm exposure, and reaching the maximum in 4 days. At 2.5 ppm of Cl2, tolerance was significant only after 10 days of exposure. Rats tolerant to Cl2 also showed cross-tolerance to HCHO. Tolerance to HCHO was observed in rats exposed to 28 ppm for 4 days, but not in groups exposed to 15 ppm for 1, 4, or 10 days. However, significant cross-tolerance to Cl2 was evident following a 1-day exposure to 15 ppm HCHO, with greatest effect seen in the group exposed for 10 days. Tolerance was reduced after a 7-day recovery following a 4-day exposure. Cross-tolerance was reduced also, but to a much lesser extent. These results suggest a common mechanism for tolerance and cross-tolerance development, but different reactive sites may exist for Cl2 and HCHO at the trigeminal nerve endings.

Animals↗

Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde.

A series of immune function and host resistance parameters were examined in female B6C3F1 mice following a 21-day (6 hr/day) inhalation exposure to 15 ppm of formaldehyde (HCHO). Immune parameters examined included delayed hypersensitivity to keyhole limpet hemocyanin, antibody plaque-forming cell response to sheep erythrocytes (T-lymphocyte-dependent antigen) and TNP-Ficoll (T-lymphocyte-independent antigen), lymphoid organ weights and histopathology, routine hematology, bone marrow cellularity and CFU progenitor cell enumeration, lymphocyte subpopulation quantitation by cell surface markers, mitogen-induced lymphocyte blastogenesis, macrophage function parameters, and host resistance to challenge with the bacterium Listeria monocytogenes and transplantable tumor cells. Lymphoid organ weight, bone marrow cellularity, and hematology parameters were unchanged in HCHO exposed mice. Similarly, the percentage of T and B lymphocytes and their proliferative responses to mitogens were not significantly altered. Antibody (IgM) plaque-forming cell response following antigen challenge was unchanged. Macrophage function was normal although some evidence of enhanced H2O2 production associated with elevated bactericidal activity was observed in resident macrophages. Resistance to challenge with the bacteria Listeria monocytogenes was significantly enhanced, while resistance to tumor challenge remained unchanged. No evidence of immunosuppression following short-term exposure to HCHO was observed.

Animals↗

Acute exposure to formaldehyde induces hepatic metallothionein synthesis in mice.

Humans risk inadvertent intraperitoneal or intravenous exposure to formaldehyde (HCHO), commonly used for disinfection of implanted or extracorporeal medical devices. Various chemical and physical stresses are known to induce hepatic metallothionein. This study examined the effect of acute parenteral administration of HCHO on induction of hepatic metallothionein synthesis. Adult male CF1 mice were administered HCHO ip and hepatic metallothionein was quantified by the cadmium-radioassay method. HCHO (50 mg/kg) increased hepatic metallothionein as early as 8 hr after dosing with maximal levels (27-fold increase) occurring at 72 hr. Metallothionein concentrations were elevated (15-fold) 24 hr after 50 or 100 mg HCHO/kg but not at lower dosages. Concomitant elevations in hepatic zinc and copper content were observed. No increases in metallothionein were observed in kidney, pancreas, or intestine 24 hr after HCHO administration (100 mg/kg, ip). Induction of metallothionein by HCHO may reflect direct de novo synthesis since the response was abolished by pretreatment with the RNA synthesis inhibitor, actinomycin D. HCHO induction of metallothionein also does not appear to be mediated by stress-induced release of corticosteroids or catecholamines from the adrenal since the response was unaltered in adrenalectomized mice. Interference with the glutathione (GSH)-dependent oxidation of HCHO by reducing hepatic GSH concentrations to 40% of control after a 2-hr pretreatment with phorone decreased the metallothionein induction response to HCHO by 33%. This result suggests that the induction may be partially due to a HCHO metabolite, e.g., formate. Confirmation of metallothionein synthesis was obtained following spectral and chromatographic analysis. Thus, HCHO and/or a metabolite produces a marked increase in hepatic metallothionein and alters hepatic zinc and copper homeostasis, all of which are transient responses. Although HCHO was only mildly hepatotoxic at the highest dose (as evidenced by an increase in plasma alanine aminotransferase activity), such changes in metallothionein synthesis and essential metal homeostasis may be part of a cellular repair mechanism operant after acute toxic cell injury.

Animals↗

Inhibition and recovery of mammalian respiratory ciliary function after formaldehyde exposure.

Formaldehyde (HCHO) has been reported to impair mucociliary clearance. The present investigation using rabbit and porcine tracheal explants in vitro examined (1) the impairment of ciliary activity, an essential component of mucociliary clearance; (2) the reversibility of ciliary dysfunction after HCHO exposure; and (3) the mechanism by which ciliary activity is reduced by HCHO. HCHO treatment of rabbit tracheal rings significantly decreased zones of active ciliated epithelium in direct proportion to concentration and exposure duration. There was also a significant concentration-dependent reduction of ciliary beat frequency. Removal of HCHO permitted recovery of zones of ciliary activity to normal beat frequencies; greater inhibitory concentrations of HCHO required greater time for return of function. Treatment of porcine tracheae with increasing concentrations of HCHO for time periods inhibitory to rabbit ciliary activity correspondingly reduced the yield of cilia extractable from treated epithelium. Furthermore, the specific activity of ATPase of extracted ciliary axonemes was diminished with increasing HCHO concentration, indicating loss of function. A recovery period following identical exposures of the porcine tracheae to the lower HCHO concentrations resulted in normal yields of functionally intact ciliary axonemes. Similarly, a recovery period after the highest HCHO concentration produced more functional axonemes than obtained from exposed tracheae without a recovery period, although less than normal yields. Therefore, ciliary dysfunction elicited by a defined range of HCHO concentrations is reversible. The yield and functional integrity of ciliary axonemes from epithelium exposed to HCHO with a recovery period are significantly greater than those without such a recovery period, suggesting an alteration and subsequent repair of epithelial surface components following HCHO exposure.

Adenosine Triphosphatases↗

Dichloromethane (methylene chloride): metabolism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice and Syrian golden hamsters.

Dichloromethane (DCM) is metabolized via a glutathione transferase (GST)-dependent pathway to formaldehyde (HCHO), a mutagenic compound that could play an important role in the carcinogenic effects of DCM observed in the liver and lungs of B6C3F1 mice at 2000 and 4000 ppm. Syrian hamsters metabolize DCM more slowly than mice via this pathway, and hamsters exposed to 3500 ppm showed no apparent carcinogenic response. The possible formation of DNA-protein cross-links (DPX) from DCM in both species was examined. Male mice and hamsters were pre-exposed for 2 days (6 hr/day) to 4000 ppm of DCM and on the third day were exposed (6 hr) to a decaying concentration (4500 to 2500 ppm) of [14C]DCM. DPX were detected in mouse liver, but not in mouse lung, hamster liver, or hamster lung. The failure to detect DPX in mouse lung does not exclude their possible formation in a subpopulation of lung cells. Metabolic incorporation of 14C derived from [14C]DCM into DNA suggested a higher rate of turnover of some mouse lung cells than of hamster lung cells, but no large difference in the turnover rates of liver cells in the two species under these conditions. These results demonstrate that HCHO derived from DCM can form DNA-protein cross-links in the liver of the B6C3F1 mouse. The formation of DPX is dependent on the activity of the GST pathway, and species such as hamsters and humans having much lower rates of DCM metabolism via this pathway may not generate toxicologically significant concentrations of HCHO and DPX.

Animals↗

Behavioral alterations induced by formaldehyde-derived tetrahydroisoquinolines.

Rats administered intracerebroventricular (IVT) injections of 3-Carboxy-1,2,3,4-tetrahydroisoquinoline (I) or 6,7-Dihydroxy-1,2,3,4-tetrahydroisoquinoline (II), the condensation products of formaldehyde with phenylalanine and dopamine, respectively, were evaluated in the open field and shuttle box situation. In the open field, 45 min after the acute administration of I, several repetitive purposeless responses and other manifestations such as teeth chattering, wet dog shakes and stiffness of the tail were observed; however, no quantitative effects were detected in the behavioral parameters recorded. Further studies were performed utilizing the shuttle box situation with drugs I and II. They produced an increase in the number of escapes and latency of shocks when measured 45 min after IVT administration (10 mg/ml and 1 mg/ml, respectively; 20 microliters). Our data suggest that the studied compounds induce qualitative alterations as well as changes in shuttle avoidance behavior in the rat when measured 45 min after administered. The effects were found to be dose and time dependent, being reversible when measured 24 and 48 hours after administration of the drugs. The present results open the possibility that the in vivo formation of tetrahydroisoquinolines could be involved in some mental pathologies, such as schizophrenia, as has been previously suggested.

Animals↗

Anaphylactic shock caused by formaldehyde in a patient undergoing long-term hemodialysis.

Formaldehyde (F) elicits contact dermatitis and asthma in subjects who are exposed to this chemical when it is applied on the skin or inhaled. An immediate-type hypersensitivity reaction was never confirmed in these reactions. F is widely used in hemodialysis to sterilize adhesives, surgical devices, or reconditioned dialyzers. A 20-year-old woman who was subjected to hemodialysis for the past 4 years had a contact dermatitis to F. When hemodialysis was performed with a new dialyzer not sterilized with F, there were no symptoms. She had minor symptoms of anaphylaxis characterized by rhinitis, wheezing, and headache on the first use of a reconditioned dialyzer. Two days later, she was dialyzed with the same reconditioned dialyzer and developed within minutes a severe anaphylactic shock requiring resuscitation. The patient had no personal or family history of atopy. Prick tests and RAST to common food and inhlant allergens were negative. Prick tests performed with 0.1% and 1% F were positive in the patient, whereas they were negative in control subjects. RAST to F was performed with discs specially prepared and coated with human serum albumin. RAST was strongly positive. RAST to ethylene oxide was negative. A patch test with F was performed and induced an anaphylactic shock 26 hours after the skin application of F. The patient did not present any anaphylactic symptoms with the use of nonreconditioned dialyzers. An immediate-type allergy to F mediated by IgE may be envisaged in this patient.

Adult↗

IgG antibody against formaldehyde human serum proteins: a comparison with other IgG antibodies against inhalant proteins and reactive chemicals.

Immune responses to formaldehyde (F) have been recorded for seven decades. More recently, sensitive assays for antibody against F-human serum albumin (HSA) have been reported. IgG antibody against F-HSA has been said to correlate with symptoms against F-HSA. We report on 61 serum samples analyzed for IgG antibodies against F-HSA. IgG antibodies against F-HSA were most prevalent in subjects who had received intravenous F. In no case (either presumed symptomatic to F or with IgG antibody against F-HSA) was there a correlation of serologic results with symptoms. We also reviewed inhalation disease caused by chemicals and proteins acting as immunogens and report that at this time there is no evidence that gaseous F meets the criteria for causation of inhalational IgG-mediated lung disease by clinical or serologic studies. Very high IgG antibody levels occur in respiratory immunologic inhalational disease, and the absence of these high IgG levels against F is strong evidence against F or F proteins being an inhalational antigen of significance.

Allergens↗

Identification of specific crosslinked histones after treatment of chromatin with formaldehyde.

Treatment of chromatin or nuclei with formaldehyde or glutaraldehyde results in the appearance of crosslinked histones as analyzed on SDS gels. The identity of two major crosslinked products was determined by iodinating the product and analzying the tryptic peptides. The results show that one product is a dimer of histones IIb2 and IIb1 and another product is a dimer of histones IIb2 and IV. These species represent no more than 15% of the total cellular histone.

Animals↗

Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent.

A new procedure is described which allows selective reversal of formaldehyde cross-linking in both histone-histone and histone-DNA of nuclei isolated from calf thymus. All ten possible dimers of the four non-H1 histones, H3, H2B, H2A and H4, are observed, the major dimers being H3-H3, H3-H2A, H2B-H2A, H2a-H2A and two separate dimers of H2B-H4. Although oligomers of the non-H1 histones are formed by prolonged treatment with this reagent, 50% of the histones continue to remain resistant to cross-linking with each other. For those histones which cross-linking with each other. For those histones which cross-link, the site of cross-linking within the molecules is located in the "core" (trysin-resistant) regionand therfore indicates proximities for these molecules within the nucleosome. The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA.

Binding Sites↗