Sensitizing Potential of Chromated Copper Arsenate in Local Lymph Node Assays Differs with the Solvent Used

Abstract

We used the local lymph node assay to evaluate the abilities of chromated copper arsenate (CCA), a commonly used wood preservative, and its components to cause sensitizing reactions after their dilution in acetone-olive oil (AOO) and dimethyl sulfoxide (DMSO). After CBA/J mice were treated topically with 0.3 to 10% CCA, 0.3–3% chromium oxide, 0.3–3% arsenic oxide, or 0.3–3% copper oxide, their auricular lymph nodes (LN) were weighed and used in lymphocyte proliferation assays. In addition, total levels of chromium and arsenic in blood samples were measured. In all groups treated with CCA in AOO or DMSO, all parameters, including LN weight and lymphocyte proliferation, increased in a dose-dependent manner. The stimulation index (SI; the mean [³H]-TdR incorporation of the treatment group divided by that of the control group) showed a positive response (SI > 3) in all treatment groups; the EC3 values (estimated concentration to yield SI of 3) of CCA in AOO and DMSO were 1.86% and < 0.3%, respectively. In addition, we confirmed that the three components of CCA - chromium oxide, arsenic oxide and copper oxide—each individually exerted sensitizing ability. Mice treated with arsenic oxide in AOO or DMSO yielded nearly equal positive responses; however, the LLNA responses in mice treated with chromium oxide and copper oxide was much higher in the DMSO groups than in the AOO groups. The total chromium level in blood was higher in DMSO groups than AOO groups, whereas arsenic levels were comparable between the DMSO and AOO groups. Our findings suggest that CCA has sensitizing activity and that the type of solvent used can influence the results of sensitization assays evaluating metals.

Keywords :

• chromated copper arsenate (CCA)
• chromic oxide
• copper oxide
• arsenic oxide
• local lymph node assay

INTRODUCTION

Since the 1960s, chromated copper arsenate (CCA) has been one of the most commonly used wood preservatives worldwide (Lebow, 1996; Solo-Gabriele and Townsend, 1999; American Wood Preservers Associations, 2005; Kim et al., 2007; Shibata et al., 2007). CCA is water-soluble and is introduced under high pressure into the pores of wood to prevent decay caused by bacteria, fungi, or insects (Warner and Solomon, 1990; Weis and Weis, 1994). The availability of CCA led to its wide use to treat wood intended for outdoor structures, such as playsets, decks, fences, utility poles, and marine docks, in many countries (Lebow, 1996; Solo-Gabriele and Townsend, 1999; American Wood Preservers Association, 2005; Shibata et al., 2007). However, the heavy metal in CCA diffuses out of, and is eluted from, treated wood during its processing for disposal (Townsend et al., 2004; Jambeck et al., 2006).

Recently, CCA was the subject of risk assessments by the United States Environmental Protection Agency (EPA) and U.S. Consumer Product Safety Commission (CPSC) for potential exposure of children that come into contact with CCA-treated playsets and home decks (U.S. EPA, 2001; Dang et al., 2003; U.S. CPSC, 2003; Zartarian et al., 2006). In response to these risk assessments, manufacturers of CCA voluntarily began to develop alternative wood preservatives, and as of 1 January 2004 CCA-treated wood has no longer been manufactured for residential use in the United States (U.S. EPA, 2002). However, although CCA-treated wood is no longer available for new construction, many CCA-treated structures still exist, and this chemical can persist in the wood for 10 to 40 years (Lebow, 1996; McQueen and Stevens, 1998; Hingston et al., 2001; Alderman et al., 2003). Therefore, the possibility of current and future exposure of humans to toxic metals from CCA-treated wood remains.

Although each of the three chemicals in CCA individually is toxic to the gastrointestinal, nervous, circulatory, and immune systems (Burrows, 1983; Sikorski et al., 1989; Cuzick et al., 1992; Tamaki and Frankenberger, 1992; Rahman et al., 1996; Flora et al., 1998; Abernathy et al., 1999; Ryan et al., 2000; Hughes, 2002; Iyer et al., 2002), the overall toxicity of CCA remains unknown. In particular, we are interested in elucidating the overall toxicity of CCA to the immune system, especially the allergenicity of total CCA. In many countries, metals are the most common causes of allergic contact dermatitis (Cronin, 1980; Bock et al., 2003; Goon and Goh, 2005), and chromium and copper allergens are present in almost all standard patch test series worldwide (Basketter et al., 1993, 2003; Bock et al., 2003; Goon and Goh, 2005; Hostynek and Maibach, 2004). However, the measured sensitizing property of metals can vary among the detection methods and solvents used.

We used the local lymph node assay (LLNA) (National Institute of Environmental Health Sciences, 1999; Basketter et al., 2002) to examine the sensitization reactions caused by chromated copper arsenate (CCA) and its components in two different solvents, acetone-olive oil (AOO) and dimethyl sulfoxide (DMSO). The LLNA was developed initially for hazard identification (Kimber and Basketter, 1992; Basketter et al., 1996, 1999; Dearman et al., 1999; Kimber et al., 2003) and has now been evaluated extensively and validated formally (Kimber and Basketter, 1992; Dearman et al., 1999; Gerberick et al., 2000; Basketter et al., 2002; Kimber et al., 2003).

MATERIALS AND METHODS

Chemicals and Reagents

Three CCA formulations, referred to as types A, B and C have been developed (Hingston et al., 2001). The CCA type B mixture we used comprised arsenic (V) oxide (45.1% as As₂O₅, w/w), chromium (VI) oxide (35.3% as CrO₃, w/w), and copper (II) oxide (19.6% as CuO₂, w/w) (American Wood Preservers Associations, 2005). Arsenic oxide was purchased from Kishida Chemical Co. (Osaka, Japan). Chromium oxide and copper oxide were purchased from Kanto Chemical Co. (Tokyo, Japan). Trimellitic anhydride (TMA) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). The maximum doses used in this study were selected to avoid systemic toxicity and/or excessive local sensitization (particularly in the preliminary test) while still permitting comparisons of the sensitizing potencies among the chemicals to be made. Test chemicals were dissolved in AOO (4:1) and DMSO, as described in Table 1.

TABLE 1 Vehicles and concentrations used in this test

Compound	Vehicles	Concentrations (%)
CCA	AOO, DMSO	0.3, 1, 3, 10
CrO3	AOO, DMSO	0.3, 1, 3
As2O5	AOO, DMSO	0.3, 1, 3
CuO2	AOO, DMSO	0.3, 1, 3
TMA	AOO	0.1, 0.3, 1, 3

AOO, acetone-olive oil (4:1); As₂O₅, arsenic (V) oxide; CCA, chromated copper arsenate; CrO₃, chromium (VI) oxide; CuO, copper (II) oxide; DMSO, dimethyl sulfoxide; TMA, trimellitic anhydride.

Animals and Housing Conditions

Female CBA/J mice (age 7-wk) purchased from Charles River Japan Laboratories (Atugi, Kanagawa, Japan) were housed individually in rooms under controlled conditions of lighting (lights on from 0700 to 1900 hr), temperature (22 ± 2°C), humidity (55% ± 15%), and ventilation (at least ten 100% fresh air changes hourly). Food (Certified Pellet Diet MF, Oriental Yeast Co., Tokyo, Japan) and water were available ad libitum. This study was conducted in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science (JALAS, 1987).

EXPERIMENTAL DESIGN

After a 1-wk acclimation period, mice were allocated randomly to dose and control groups (n = 5 per group). A 25 μ l aliquot of test solution or solvent only was applied daily to the dorsum of each ear of each mouse for three consecutive days (Days 1 through 3). On Day 6, [³H]-methyl thymidine ([³H]-TdR, 20 μCi/animal, GE Healthcare Bioscience, Tokyo, Japan) was injected via the tail vein into all test and control mice. Five hours after the injection, the mice were euthanized, and each animal's auricular lymph nodes (LN) were removed, weighed, and pooled in PBS (Gibco, Tokyo, Japan).

ATP Activity

Single-cell suspensions of LN in 5 ml PBS were prepared by passage through sterile 70-μ m nylon cell strainers (Falcon, Tokyo, Japan); 100 μ l of each cell suspension was seeded in duplicate into opaque-walled 96-well plates (Nulge Nunc International, Tokyo, Japan). The ATP in each well was measured with a luciferin–luciferase system (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Tokyo, Japan). The ATP activity (in relative light units [RLU]) was determined with a microplate luminometer (TR717, Berthold Japan, Tokyo, Japan).

Incorporation of [³H]-TdR

The remaining LN cell suspension was washed twice with an excess of PBS, and precipitate was incubated in 3 ml of 5% trichloroacetic acid (TCA, Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 4°C for ≈18 hr. Each cell pellet was resuspended in 1 ml TCA and transferred to 9 ml of scintillation fluid (AtomLight, PerkinElmer Japan, Tokyo, Japan). Incorporation of [³H]-TdR was measured by β -scintillation counter (LC-5100, Aloka, Tokyo, Japan) as disintegrations per minute (dpm) for each mouse and expressed as dpm/mouse.

Analysis of Total Chromium and Arsenic in Blood

Blood samples from two mice treated with solvent only (AOO or DMSO) or chromium oxide (3% with AOO or DMSO) or arsenic oxide (3% with AOO or DMSO) were collected 5 hr after the last dose of chemical and stored at −70°C until analysis. Total chromium and arsenic were measured by inductively coupled plasma mass spectrometry (Agilent 7500c ICP-MS, Agilent Technologies, Santa Clara, CA) and atomic absorption photometry with hydride generation (Spectr AA220, Varian, Palo Alto, CA), respectively. For analyzing chromium, yttrium was used as an internal standard.

Evaluation of LLNA Data

Stimulation indexes (SIs) and EC3 values were calculated by using the [³H]-TdR incorporation data. The SI was calculated by dividing the mean [³H]-TdR incorporation value for each treatment group by that of the solvent control group. The EC3 value is an estimate of the amount of the test solution required to induce an SI of 3 (Basketter et al., 1999). In the standard LLNA, the criterion for a positive response is an SI of 3 or greater (Kimber and Basketter, 1992; Dearman et al., 1999). Data on LN weights, ATP activity, and incorporation of [³H]-TdR were compared by Dunnett's multiple comparison test and significant differences were considered at p < 0.05.

RESULTS

The LLNA results for the various CCA and its components treatment groups are presented in Table 2, Table 3, and Table 4. When CCA was dissolved in AOO, LN weights and measures of lymphocyte proliferation, including ATP activity and incorporation of [³H]-TdR, increased in a dose-dependent manner, and differences between test and control values achieved statistical significance in the 3% and 10% treatment groups. In the context of [³H]-TdR incorporation, CCA induced a positive response, defined as an SI of 3 or greater, in the 3% and 10% treatment groups. In comparison, CCA in DMSO significantly increased LN weight and lymphocyte proliferation in the 1%, 3%, and 10% dose groups, and 0.3% CCA induced a positive response in regard to [³H]-TdR incorporation. The EC3 value of CCA in AOO was 1.86% and that for CCA in DMSO was < 0.3% (Table 4). The relative skin sensitization potencies based on derived EC3 values (Kimber et al., 2003) indicate that CCA in AOO is a moderate sensitizer and CCA in DMSO is a strong sensitizer.

TABLE 2 Results of LLNA performed with CCA and TMA

				[^3H]-TdR incorporation
Chemical	Dose	Lymph node weight	Lymphocyte proliferation	dpm
(vehicle)	(%)	(mg; mean ± 1 SD)	(RLU; mean ± 1 SD)	(mean ± 1 SD)	SI^a
CCA (AOO)	0	4.08 ± 0.68	172409 ± 26525	255 ± 44	1.00
	0.3	4.96 ± 1.06	210227 ± 33755	398 ± 67	1.56
	1	4.96 ± 0.53	230195 ± 22439	453 ± 76	1.78
	3	6.18 ± 1.57	**361201 ± 59859	**1181 ± 232	4.63
	10	**9.76 ± 1.86	**682681 ± 127078	**3497 ± 908	13.71
CCA (DMSO)	0	4.58 ± 0.79	208154 ± 58985	879 ± 259	1.00
	0.3	6.26 ± 0.45	434494 ± 129186	3267 ± 875	3.72
	1	**10.70 ± 1.15	**753236 ± 159248	**11337 ± 3844	12.90
	3	**15.88 ± 3.19	**1106000 ± 228346	**24339 ± 6143	27.69
	10	**12.36 ± 2.48	**743042 ± 162309	**18429 ± 4012	20.97
TMA (AOO)	0	4.73 ± 0.29	107154 ± 23141	190 ± 48	1.00
	0.1	5.53 ± 0.50	193606 ± 79122	602 ± 321	3.17
	0.3	7.83 ± 0.87	252483 ± 76540	1493 ± 783	7.86
	1	**11.20 ± 1.80	418576 ± 73060	**4480 ± 1117	23.60
	3	**17.30 ± 1.47	**916892 ± 197348	**11988 ± 1474	63.10

^aSI is the ratio of the mean of the measurements of the treatment group to that of the vehicle control group. The criterion for a positive response in the standard LLNA was an SI of 3 or greater. Values for treatment and control groups were compared by Dunnett's multiple comparison test. * p < 0.05; ** p < 0.01.

TABLE 3 Results of LLNA performed with components of CCA

				[^3H]-TdR incorporation
Chemical	Dose	Lymph node weight	Lymphocyte proliferation	dpm
(vehicle)	(%)	(mg; mean ± 1 SD)	(RLU; mean ± 1 SD)	(mean ± 1 SD)	SI^a
CrO3 (AOO)	0	4.38 ± 0.74	123888 ± 46240	204 ± 112	1.00
	0.3	5.86 ± 1.22	157784 ± 34049	339 ± 59	1.66
	1	5.56 ± 0.59	**207682 ± 41551	373 ± 79	1.83
	3	5.32 ± 1.07	**210115 ± 26502	607 ± 354	2.98
CrO3 (DMSO)	0	5.20 ± 0.91	127896 ± 46209	457 ± 178	1.00
	0.3	**11.15 ± 2.79	**513344 ± 127789	9426 ± 3257	20.63
	1	**16.33 ± 2.54	**740568 ± 157185	**19454 ± 5568	42.57
	3	10.73 ± 2.13	333636 ± 58636	8489 ± 4772	18.58
As2O5 (AOO)	0	4.82 ± 1.33	161335 ± 48109	232 ± 74	1.00
	0.3	6.04 ± 1.16	242568 ± 72220	398 ± 144	1.72
	1	**8.54 ± 0.66	**369757 ± 35344	**816 ± 106	3.52
	3	**8.58 ± 2.30	**395299 ± 103007	**2001 ± 486	8.63
As2O5 (DMSO)	0	5.20 ± 0.91	127896 ± 46209	457 ± 178	1.00
	0.3	7.63 ± 2.20	213730 ± 46714	1744 ± 1783	3.82
	1	8.43 ± 1.26	277102 ± 99223	1650 ± 1140	3.61
	3	**9.28 ± 0.93	**324468 ± 78410	1919 ± 442	4.20
CuO2 (AOO)	0	4.84 ± 0.91	100997 ± 33211	204 ± 96	1.00
	0.3	5.32 ± 1.14	148323 ± 35002	244 ± 51	1.20
	1	4.36 ± 0.73	125780 ± 32725	256 ± 120	1.25
	3	6.02 ± 0.39	**200666 ± 45797	**511 ± 220	2.50
CuO2 (DMSO)	0	5.20 ± 0.91	127896 ± 46209	457 ± 178	1.00
	0.3	5.03 ± 0.51	197392 ± 28422	795 ± 137	1.74
	1	5.80 ± 1.57	258838 ± 76667	932 ± 382	2.04
	3	7.27 ± 0.21	**335755 ± 23020	**2209 ± 623	4.83

^aSI is the ratio of the mean of the measurements of the treatment group to that of the vehicle control group. The criterion for a positive response in the standard LLNA was an SI of 3 or greater. Values for treatment and control groups were compared by Dunnett's multiple comparison test. * p < 0.05; ** p < 0.01.

TABLE 4 EC3 values

	EC3 value (%)
Group	AOO	DMSO
CCA	1.86	< 0.3
CrO3	−	< 0.3
As2O5	0.80	< 0.3
CuO2	−	1.69
TMA	< 0.1	not done

−, SI was less than 3.

EC3 value was an estimation of the amount of the test solution required in LLNA to induce a SI of 3.

We then investigated the sensitizing properties of the individual components of CCA (Table 3). When DMSO was the solvent, lymphocyte proliferation was increased significantly after treatment of mice with 0.3% chromium oxide (Cr), 1% arsenic oxide (As), or 3% copper oxide (Cu). When AOO was the solvent, lymphocyte proliferation was increased significantly at the 1% dose level for all components. Further, Cr and Cu at 0.3% and 3% in DMSO, respectively, generated positive responses based on the SI, leading to EC3 values of < 0.3% and 1.69%, respectively (Table 4). Cr and Cu in DMSO were categorized as extreme and moderate skin sensitizers, respectively. However, neither Cr nor Cu in AOO stimulated a positive response according to SI results, regardless of dose.

Arsenic oxide at 1% in DMSO and 0.3% in AOO led to positive SI responses, correlating with EC3 values of 0.80% for As in AOO and < 0.3% for As in DMSO. Arsenic oxide was a relatively strong skin sensitizer regardless of the solvent used. Throughout the LLNA of CCA and its components, ATP activity tended to increase in parallel with [³H]-TdR incorporation. In concurrent positive controls, TMA induced an extreme sensitizing response (EC3, < 0.1%).

Because the absorption of CCA and its components through the skin is influenced by the solvent in which they are dissolved, the results of LLNA can vary with the solvent choice. To clarify the difference in absorbability between AOO and DMSO, we measured the total levels of chromium and arsenic in blood by using inductively coupled plasma mass spectrometry and atomic absorption photometry with hydride generation, respectively (Table 5). These chemical analyses revealed that the total blood chromium level was higher in DMSO groups than AOO groups, whereas arsenic levels were comparable between DMSO and AOO groups. These results demonstrate that solubility and dermal penetration of a compound—and thus the results of LLNA—vary according to the solvent used.

TABLE 5 Chromium oxide and arsenic oxide levels in blood

		Mean absorption (mg/kg)
Group	Vehicle	CrO3	As2O5
Control	AOO	< 0.1	< 0.1
	DMSO	< 0.1	< 0.1
CrO3	AOO	0.52	not done
	DMSO	2.00	not done
As2O5	AOO	not done	1.25
	DMSO	not done	0.76

Blood samples from two mice treated with solvent only (AOO or DMSO) or chromium oxide (3% with AOO or DMSO) or arsenic oxide (3% with AOO or DMSO) were collected 5 hr after the last dose of chemical. Total chromium and arsenic were measured by inductively-coupled plasma mass spectrometry and atomic absorption photometry with hydride generation, respectively.

DISCUSSION

Chromated copper arsenate (CCA)-treated wood has been phased out for residential applications, but many CCA-treated building outdoor structures, such as decks, docks, and playgrounds, still exist. In 1970, the total volume of treated wood products was 248 million cubic feet, of which 39 million cubic feet was treated with CCA. The total rose to 591 million cubic feet in 1996, with 467 million cubic feet of it CCA-treated products (Holton, 2001). Especially, children are at high risk of being sensitized to CCA by percutaneous absorption. The Consumer Product Safety commission (CPSC) conducted a series of tests to gauge the risks to children of contact with CCA-treated play sets and published the results in the 1990 report. The CPSC found that the risk of skin cancer through ingestion of CCA dislodged from playground equipment ranged from less than one in a million to nine in a million (Fields, 2001). We thought that the same might hold true with regard to contact hypersensitivity.

Our results suggest that the LLNA method reveals the skin sensitization potential of CCA as well as its compounds reflecting its individual components (see Table 2, Table 3, Table 4). Contact allergens can be categorized on the basis of relative skin sensitization potency based on derived EC3 values (Kimber et al., 2003). Accordingly, CCA in AOO was categorized as a moderate sensitizer and CCA in DMSO as a strong sensitizer. According to the above, the results differed somewhat depending on the solvent in which the test chemical was dissolved (Table 2, Table 3, Table 4, Table 5). These findings clarify the importance of choosing appropriate solvents for LLNA, and important factors for consideration are the solubility and dermal penetration of the target compound in the selected solvent. Metals and metal salts are highly soluble in DMSO. In addition, as a penetration enhancer, DMSO increases skin hydration, LN effect due to irritation and thus promotes diffusion of hydrophilic components (Robinson and Sozeri, 1990; Ikarashi et al., 1993; Ryan et al., 2002).

In the case of several metals (Basketter et al., 1999; Hostyneck and Maibach, 2004), repeated application of DMSO increases the sensitivity of the LLNA. In general, an enhancing effect of DMSO on skin penetration has been demonstrated experimentally in guinea pigs in vivo (Wahlberg and Skog, 1967; Mor et al., 1988). In this study, when CCA and its components were dissolved in AOO, arsenic oxide emerged as a key contributor to the overall sensitizing property of CCA. In contrast, all three components of CCA induced positive sensitization responses when the solvent used was DMSO. Arsenic oxide was a relatively strong skin sensitizer regardless of the solvent used. LLNA responses and arsenic levels in blood were comparable between DMSO and AOO groups. This suggested that the enhancing effect of DMSO might not necessarily be consistent for all metals/metal compounds.

In this study, we confirmed that the three components of CCA—chromium oxide, arsenic oxide, and copper oxide—each individually exerted sensitizing ability. Various chromium- and copper-containing compounds (e.g., sulfate, sodium, and chloride salts) have already been evaluated for their sensitizing properties in the guinea pig test (maximization test and occluded patch test of Buehler)—a standard method used as predictor of skin sensitization potential (Magnusson and Kligman, 1969)—and the LLNA (Boman et al., 1979; Cronin, 1980; Burrows, 1983; Fisher, 1986; Fowler, 1990; Robinson and Sozeri, 1990; Ikarashi et al., 1992, 1996; Basketter et al., 1993, 1996, 1999, 2003; Rycroft et al., 1995; Kimber et al., 1996, 2003; Iyer et al., 2002; Bock et al., 2003; Hostynek and Maibach, 2004; Makinen and Linnainmaa, 2004). According to these reports, various chromium- and copper-containing compounds significantly increased lymph node cell proliferation.

Unlike for chromium and copper, the sensitizing properties of various arsenic-containing compounds have not yet been tested in detail. Arsenic, a member of the Group VA elements of the periodic table, can naturally exist in the +5, +3, 0, and −3 oxidation states (Cai and DuBow, 1997; Wang et al., 2006). In CCA, the form of arsenic present is arsenate, a pentavalent form that is chemically similar to a phosphate ion. In general, the toxicity of arsenicals depends on their bioavailability, oxidation state, and their physicochemical form (i.e., organometalloid, inorganic salt, etc.; Tamaki and Frankenberger, 1992; Cai and DuBow, 1997). Among its best-known toxicities, inorganic arsenic has been shown to act as a key etiologic component in the development of peripheral vascular disease and skin, lung, liver, bladder, kidney, and prostate cancers (Cuzick et al., 1992; Wingren and Axleson, 1993; Christensen and Poulsen, 1994; Rahman et al., 1996; Abernathy et al., 1999; Ryan et al., 2000; Patterson et al., 2004).

To our knowledge, ours is the first report to demonstrate the sensitizing property of select arsenic-containing compounds. Thus far, inorganic arsenic has not been believed to penetrate intact skin, and contact exposure to arsenic-contaminated water is not considered a serious health risk (Lansdown, 1995; Hall, 2002). However, the present study clearly demonstrated that arsenic oxide induced a positive response in the LLNA and led to high arsenic levels in blood.

To date, the relationship between arsenic oxide exposure and allergenicity remains poorly understood. In several reports, arsenic-induced stimulation of immune-related endpoints has appeared to parallel a suppression of immune functions (Sikorski et al., 1989; Flora et al., 1998; Institoris et al., 2001). In vitro, trivalent arsenicals acted to up-regulate expression of several allergy-related cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-6, and GM-CSF) in kerationocytes (Germolec et al., 1996, 1997, 1998; Gaspari, 2007). Similarly, sodium arsenate stimulated synthesis of IL-1α in HEL30 murine kerationcytes (Corsini et al., 1999; Corsini and Galli, 2000). Furthermore, Patterson et al. (2004) demonstrated that arsenic altered the secretion of growth promoting and inflammatory cytokines that can regulate Langerhans cell migration and maturation during allergic contact dermatitis. This is because: (A) allergic contact hypersensitivity is a delayed-type reaction during which Langerhans cells recognize a foreign antigen, transport it to the local lymph nodes, and present it to T-lymphocytes (which subsequently undergo clonal proliferation); and, (B) Langerhans cell migration and maturation is mediated, in part, by keratinocyte-derived cytokines (Corsini and Galli, 2000), this suggests to us that growth factor secretion induced by arsenic agents could be accompanied by the appearances of both an increased allergenicity and of LN cell proliferation.

An additional mechanism by which the arsenicals could impact on allerginicity is based on the fact that select metal compounds can act as haptens that bind to proteins or to a cell membrane; in so doing, these agents gain a sensitizing ability. Many studies (Picardo et al., 1990; Ikarashi et al., 1992; Basketter et al., 1999; Bock et al., 2003; Hostynek and Maibach, 2004; Goon and Goh, 2005) have attempted to elucidate the role (and associated mechanisms) of these metals in the induction of sensitization. For example, nickel ions apparently can induce an increase in the production of IL-1 by keratinocytes as well as their production of IL-2 and interferon (IFN)-γ (Picardo et al., 1990; Ikarashi et al., 1992). However, whether the same events would occur after sensitization by another metal remains uncertain. Whether arsenicals act in a manner akin to nickel, and if these nickel-like effects are related to the observations made in the current study, are currently under investigation in our laboratories.

In summary, our results suggest that the LLNA method can detect the skin sensitization potential of metals compounds, such as CCA, and its individual components. However, the LLNA results indicate that CCA in DMSO may induce stronger sensitization reactions than CCA in AOO. As such, as a cautionary note to Investigators, it should be recognized that the results of sensitization assays with metals might be greatly influenced by the selection of the solvent(s) to be used.

We thank Drs. A. Haishima, H. Fujie, and Y. Hayashi of the Institute of Environmental Toxicology for their useful discussions, suggestions, and technical assistance. This work was supported by a research Grant-in-Aid from the Ministry of Health, Labor, and Welfare of Japan.