* Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, New York 10987;
Vanderbilt School of Medicine, Memphis, Tennessee 37212; and
Toxicology Consultants, Inc., Gibsonia, Pennsylvania 15044
Received June 15, 2001; accepted January 2, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: CCA-treated wood; metals; gene expression; chromium; copper; arsenic; bioavailability; metallothionein.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data regarding the actual kinetics of release of metals from CCA wood are limited in the peer-reviewed literature, but it appears that several factors, including time, temperature, and humidity, contribute to fixation and thus the bioavailability of metals in CCA-treated wood. In freshwater environments, minimal leaching of metals from the CCA wood occurs, but is increased under acidic conditions (Warner and Solomon, 1990). In marine water environments, Weis and colleagues have demonstrated that metals are present in the fine fraction of sediments near CCA-treated bulkheads in estuaries (Weis and Weis, 1992
; Weis et al., 1998
). These metals are generally confined to within 1 m of the bulkheads and resulted in a reduction in the nearby biotic community. McNamara observed that in Southern pine wood treated with CCA, the maximum concentration of chromium that could be released from wood occurred immediately after treatment (0.27%) and diminished rapidly over a 336-h period (McNamara, 1989
). Thus, proper fixation of metals to the wood fibers after pressure treatment is essential to minimizing potential exposure of wood handlers to copper, chromium, and arsenic.
The present study is based upon the premise that the use of CCA pressure-treated wood at outdoor construction sites and in fabrication industries may result in the exposure of workers to potentially hazardous wood dust. The extent of metal exposure associated with the inspirable fraction of wood dust encountered by workers cutting, sanding, and routing CCA pressure-treated wood has been previously examined (Decker et al., 2001; Nygren et al., 1992) and, in certain job assignments, found to exceed recommended occupational exposure levels for arsenic (Decker et al., 2001). In regards to the bioavailability of metals bound to inhaled CCA wood dust, we speculate that the greater surface area of inhalable particles (relative to CCA-treated lumber), the acidic environment of phagolysosomes in phagocytic cells, and the retention time of wood particles in the respiratory tract may each contribute to an increase in bioavailability of heavy metals from CCA wood dust. Therefore, we examined the bioavailability of chromium, copper, and arsenic in vitro by examining cytotoxicity and metallothionein (MT) mRNA induction in cells treated with respirable-size particles generated by the sanding of CCA pressure-treated wood.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metal analysis.
Samples of wood dust and cell culture media were analyzed for metal content using nitric acid dissolution and inductively coupled plasma-atomic emission spectrometry (ICP-AES) as described previously (Decker et al., 2001). Briefly, samples were leached/digested in concentrated (70%) nitric acid for 5 min and then diluted and leached in a 7% nitric acid matrix for at least 24 h. The samples were then filtered with a 0.45-µm pore size filter and analyzed by IPC-AES (Thermal Jarrell Ash Polyscan 61E). The ICP was calibrated prior to sample analysis using a 7% nitric acid solution with no metals as the blank and a 7% nitric acid solution containing 5 ppm each of chromium, copper, and arsenic. Low (0.25 ppm) and high (0.5 ppm) concentration quality control samples were analyzed along with blanks during the wood dust analysis. In addition, a 5.0-ppm calibration standard was reread periodically.
Metallothionein mRNA experiments.
After treatment of V79 cells with Southern yellow pine or CCA-treated wood dust for 24 to 48 h, total cellular RNA was isolated by a rapid guanidinium-phenol extraction method using Trizol (GibcoBRL). RNA quantity and purity was determined by A260/A280 spectrophotometric absorbances. Integrity of RNA was confirmed by ethidium bromide staining of ribosomal RNA following gel electrophoresis. Standard slot-blot RNA analyses were carried out by direct application of 5 µg glyoxalated RNA samples onto nylon membrane filters (Nytran, Schleicher & Schuell, Keene, NH) in a vacuum manifold (Schleicher & Schull). Membrane-bound RNA was hybridized to nick-translated, 32P-labeled cDNA probes in the presence of dextran sulfate by a modification of the procedure of Wahl et al (1979). A rat MT-1 plasmid (Andersen et al, 1983) was provided by Dr. H. Herschman, UCLA, and the ß-actin probe was purchased from ATCC (Bethesda, MD). Following hybridization, filters were washed to a final stringency of 0.4x standard saline citrate + 0.1% sodium dodecyl sulfate at 65°C for 30 min. Specifically hybridized mRNA was visualized by film autoradiography at 70°C using Kodak XAR-5 film plus Cronex intensifying screens (DuPont, DE). Autoradiogram signal strengths of hybridized mRNA were quantitated by the measurement of optical densities using BioImager (Millipore, Ann Arbor, MI). All MT gene expression results were normalized to actin expression which served as an internal control to ensure that artifacts such as unequal loading of RNA onto filters were not responsible for any observed differences in autoradiographic signal strengths.
Collection of wood dust.
The CCA pressure-treated wood was a gift of Hickson Corporation (Conley, GA). Initially, Southern yellow pine boards (5/4" x 4" x 8`) were cut in half. One half was treated with a CCA treating solution for one full treatment cycle and the remaining half was saved as the control wood. The CCA-treated wood was wrapped in plastic, wet fixed for 7 days, and then air dried at ambient temperature. Each board was tested with chromatropic acid to ensure complete fixation. X-ray fluorescence analysis of cross sections of the treated wood demonstrated that the American Wood-Preservers' Association fixation standard of 0.4 pounds/ft2 was achieved. Upon delivery at NYU School of Medicine, the boards of CCA-treated wood and control wood were singly wrapped in autoclavable paper and autoclaved to sterilize the samples. A special sanding chamber was constructed to collect respirable size wood dust under as sterile as possible conditions for use in the in vitro experiments. A disc sander was placed in a 2 x 3 x 3` acrylic chamber supplied with HEPA-filtered air. A control board was sanded first by feeding the wood through a cut in the end of the wrapping paper into a slot in the side wall of the acrylic chamber directly opposite the sanding disc. As the wood dust particles became airborne, a cascade centripetal impactor (BGI, Inc., Waltham, MA) sampled the chamber atmosphere at 30 l/min. After appropriate sampling periods (2030 min), the centripetal sampler was opened within the sterile atmosphere of a bio-safety hood, and dust collected on the stages with effective cutoff diameters of 1.43.4 µm and less than 1.4 µm was pooled and stored in sterile test tubes at 4°C. The generation system was cleaned and the identical sanding and sampling procedure was repeated with a board of CCA-treated wood.
Statistics.
The lethal concentration of the test material which killed 50% of the colonies (LC50) was calculated manually using log-probit graph paper. Statistical comparisons of LC50 values between treatment groups were done with a Fisher's exact test. Evaluation of MT differences among treatment groups were done with a one-factor analysis of variance followed by a Student-Neumann-Keuls post hoc test at each time point. All data are presented as the mean ± SE; p values 0.05 were considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Incubation of cells with CCA-treated wood dust for 24 to 48 h produced significant increases in metallothionein mRNA. We observed these increases at least through 96 h (data not shown). Although arsenic is capable of inducing metallothionein mRNA (Albores et al., 1992; Kreppel et al., 1993
; Thornalley and Vasak, 1985
), our data suggest that copper was responsible for this induction. Incubation of the CCA-treated wood dust in pH 7.2 cell culture media for 24 h at 37°C resulted in the transfer of copper into the media (approximately 1 ppm) and no detectable As or Cr. The limit of detection by inductively coupled plasma spectrometry was 2040 ppb for the three metals. During the incubation of this extract with the cells, it is not known whether the copper was in the free state or complexed to amino acids or secreted proteins. Regardless, the copper-containing extract produced significant increases in metallothionein mRNA, thus providing strong evidence that copper was responsible for the induction of metallothionein mRNA by the CCA-treated wood dust. In terms of potential adverse health effects, the increase in metallothionein mRNA in cells incubated with CCA-treated wood dust is unclear. An increase in metallothionein protein or mRNA can occur in response to a variety of toxic agents or stressors and is often considered to be an adaptive response to the toxic effects of metals or oxidative injury (Thornalley and Vasak, 1985
; Wang et al., 1994). Regardless, our findings determined that significant amounts of copper can be transferred from CCA-treated wood dust into pH 7.2 cell culture media. Because this large degree of leaching is in sharp contrast to the relatively small amount of metals that are released from CCA-treated lumber into freshwater and marine environments, it suggests that the large surface area of respirable wood dust and mammalian body temperature may significantly increase the bioavailability of metals in fixed CCA-treated wood. These factors, as well as the phagocytic ability of pulmonary macrophages, make the inhalation of CCA-treated wood dust an occupational concern.
As determined in the colony survival bioassay, CCA-treated wood dust was more cytotoxic than the untreated Southern yellow pine wood dust generated from the same, but untreated, piece of lumber. This increase in cytotoxicity suggests that one or more of the metals becomes bioavailable during the 24-h incubation period by either release into the cell culture media or after intracellular compartmentalization of wood dust particles. Importantly, the CCA-treated wood dust maintained this greater cytotoxicity (than the control wood dust) in the arsenic-resistant subline of cells. This finding suggests that either chromium or copper was responsible for the cytotoxic effects of CCA-treated wood dust. We can further speculate that copper played little role in the cytotoxic effects of CCA-treated wood dust because the LC50 for copper chloride was approximately 150 µM (or 9 ppm), which is considerably higher than the concentration of copper in the cell culture media extract even if all of the copper was theoretically released from the CCA-treated wood dust. Because neither Cr nor As was released into the cell culture media, it is likely that the observed cytotoxicity was not due to metals that were leached from the wood dust directly into the cell culture media during the incubation period. It is more likely that the CCA-treated wood dust-induced cytotoxicity was a result of the phagocytosis of wood particles and the subsequent release of metals, presumably Cr, from lysosomal vacuoles within the cells. Because the preservative metal oxides are present in CCA-treated wood at 0.8% by weight, the equivalent EC50 of arsenate and chromate in CCA-treated wood would be approximately 5µg/ml (from Fig. 1, upper panel). Comparison of this value with the actual EC50s for chromium and arsenic as their inorganic aqueous solutions (Fig. 4
) suggests that the chromium and arsenic in CCA-treated wood may be up to 7-fold less toxic to cultured V79 cells than the inorganic water-soluble forms of these metals. Thus, bioavailability of the preservative metals in treated wood appears to play a role in the cytotoxicity of CCA-wood dust. Of course, this speculation does not take into account any potential interaction(s) of the metals that are present in combination in the CCA-treated wood.
In conclusion, these studies have demonstrated that compared with control wood dust, CCA-treated wood dust is more cytotoxic in vitro and induces an increase in the steady-state expression level of metallothionein mRNA. Thus, under the conditions of our experiments, the metals bound to wood fibers appear to become bioavailable. This bioavailability occurs despite the complete (as defined by the pressure-treated wood industry) fixation of the metals to the wood fibers. Interestingly, we observed a general decrease in cytotoxicity of the CCA-treated wood dust over a 2-month study period. This observation suggests that the freshly generated CCA-treated wood dust encountered in the workplace may represent a greater hazard than the wood dust that was used over a period of time in our bioavailability studies. Inhalation toxicology studies using respirable dust particles freshly generated from CCA-treated wood would address this issue.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albores, A., Koropatnick, J., Cherian, M. G., and Zelazowski, A. J. (1992). Arsenic induces and enhances rat hepatic metallothionein production in vivo. Chem. Biol. Interact. 85, 127140.[ISI][Medline]
American Wood Preservers Institute (1997). The 1996 Wood Preserving Industry Production Statistical Report. Fairfax, VA. Available at: http://www.preservedwood.com. Accessed February 26, 2002.
Andersen, R. D., Birren, B. W., Ganz, T., Piletz, J. E., and Herschman, H. R. (1983). Molecular cloning of the rat metallothionein 1 (MT-1) mRNA sequence. DNA 2, 1522.[ISI][Medline]
Dahlgren, S. E., and Hartford, W. W. (1972). Kinetics and Mechanism of Fixation of Cu-Cr-As Wood Preservatives, Parts I-V, Holzforschung. Technischer Verlag Herbert, Berlin.
Decker, P., Cohen, B., Butala, J. H., and Gordon, T. (in press). Exposure to wood dust and heavy metals in workers using CCA pressure-treated wood. Am. Ind. Hyg. Assoc. J.
Kreppel, M., Bauman, J. W., Liu, J., McKim, J. M., Jr., and Klaassen, C. D. (1993). Induction of metallothionein by arsenicals in mice. Fundam. Appl. Toxicol. 20, 184189.[ISI][Medline]
McNamara, W. S. (1989). CCA Fixation Experiments Parts 1 and 2. The International Research Group (IRG) on Wood Preservatives: Preservatives and Methods of Treatment. Document No. IRG/WP/3504, pp. 17. IRG Secretariat, Stockholm, Sweden.
Nygren, O., Nilsson, C. A., and Lindahl, R. (1992). Occupational exposure to chromium, copper and arsenic during work with impregnated wood in joinery shops. Ann. Occup. Hyg. 36, 509517.[ISI][Medline]
Peters, H. A., Croft, W. A., Woolson, E. A., Darcey, B., and Olson, M. (1986). Hematological, dermal and neuropsychological disease from burning and power sawing chromium-copper-arsenic (CCA)-treated wood. Acta Pharmacol. Toxicol. (Copenh). 59(Suppl.), 3943.[Medline]
Peters, H. A., Croft, W. A., Woolson, E. A., Darcey, B. A., and Olson, M. A. (1983). Seasonal arsenic exposure from burning chromium-copper-arsenate-treated wood. JAMA 251, 23932396.[ISI]
Thornalley, P. J., and Vasak, M. (1985). Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochem. Biophys. Acta 827, 3644.[ISI][Medline]
U.S. EPA. (1986). Environmental Protection Agency: Creosote, pentachlorophenol, and inorganic arsenicals; amendment of notice of intent to cancel registrations. 51 Fed. Regist., 13341348.
Wahl, G. M., Stern, M., and Stark, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. U.S.A. 76, 36833687.[Abstract]
Wang, Z., and Rossman, T. G. (1993). Stable and inducible arsenite resistance in Chinese hamster cells. Toxicol. Appl. Pharmacol. 118, 8086.[ISI][Medline]
Wang, Z., Shore, Y., and Rossman, T. G. A cadmium-sensitive Chinese hamster cell line with low constitutive level of metallothionein gene expression. In Proceedings of the First International Symposium on Metals and Genetics, Toronto, 1994, p. 26.
Warner, L. M., and Solomon, K. R. (1990). Acidity as a factor in leaching of copper, chromium, and arsenic from CCA treated dimension lumber. Environ. Toxicol. Chem. 9, 13311337.[ISI]
Weis, J. S., and Weis, P. (1992). Construction materials in estuaries: Reduction in the epibiotic community on chromated copper arsenate (CCA) treated wood. Mar. Ecol. Prog. Ser. 83, 4553.[ISI]
Weis, J. S., Weis, P., and Proctor, T. (1998). The extent of benthic impacts of CCA-treated wood structures in Atlantic coast estuaries. Arch. Environ. Contam. Toxicol. 34, 313322.[ISI][Medline]