Microscopic analysis of chromium accumulation in the bronchi and lung of chromate workers

Abstract

BACKGROUND

It is known that chromium is an inhaled carcinogen and an important risk factor in the development of lung carcinoma.

METHODS

The authors used a microscopic X-ray fluorescence analyzer with transmitted X-ray mapping imaging (Horiba, Kyoto, Japan) to measure the accumulation of chromium in 10 resected lung tissue specimens and 90 biopsy specimens from chromate workers.

RESULTS

The maximum chromium accumulation (mean ± standard deviation) in 10 resected lung tissue specimens was 197 ± 238 counts per second (cps)/mili ampere (mA) (range, 4–649 cps/mA). Chromium accumulation was scattered in six tissue specimens and diffuse in one specimen. Chromium accumulation in the proximal bronchi was less than in the bronchioles or subpleural regions of the lung. Chromium accumulation was detectable in 63 (70%) of 90 biopsy specimens, and the mean accumulation was 6.5 ± 9.2 cps/mA (range, 0–46.5 cps/mA). Chromium detected in bronchial tissue specimens was deposited in the bronchial stroma but not in the epithelium. The maximum chromium accumulations in dysplasic (n = 3), squamous metaplastic (n = 10), and normal bronchial epithelia (n = 9) in chromate workers and in normal bronchial epithelia (n = 3) in non-chromate workers were 20.2 ± 5.4, 18.3 ± 12.2, 13.2 ± 13.4, and 3.0 ± 1.8 cps/mA, respectively. The amount of chromium accumulation significantly increased according to the progression of malignant change of the bronchial epithelium (P = 0.003).

CONCLUSIONS

Previous studies found that lung carcinoma with chromate exposure exhibited a variety of genetic abnormalities. Considering genetic aberrations and chromium accumulation in these premalignant lesions is useful for elucidating the process of carcinogenesis in chromium-induced lung carcinoma. Cancer 2003. © 2003 American Cancer Society.

Lung carcinoma continues to be the leading cause of cancer-related death among men in Japan and the United States.1 Cigarette smoke is considered to be the predominant cause of lung carcinoma, particularly squamous cell carcinoma and small cell carcinoma.2 It is a heterogenous mixture of particulate and nonparticulate products from the incomplete combustion of various materials. Approximately 20 carcinogens among the multiple components of cigarette smoke are likely to be involved in lung carcinoma. The complexity of the composition of this smoke leads to some confusion about the mechanism by which it causes lung carcinoma.3

Chromium is another inhaled carcinogen that plays a significant role in lung carcinoma.4 In contrast to cigarette smoke, chromium is a unitary carcinogen, although it shows different effects according to its oxidation state.5 Most chromate particles are 1–3 μm in diameter.6 Chromium-induced lung carcinoma is clinically similar to cigarette smoke-induced lung carcinoma in four ways. First, many lung carcinoma lesions are induced in the proximal airway of the lung. Second, not only lung carcinoma but also premalignant changes (squamous metaplasia, dysplasia, and carcinoma in situ [CIS]) are induced. Third, the latent period of lung carcinoma is approximately 15–30 years. Fourth, multiple lung carcinoma lesions frequently are induced.7 To shed light on inhalation carcinogenesis, we chose to examine a population of chromate workers.

Chromium is the most extensively investigated metal with respect to mutagenicity and carcinogenicity.8 However, the deposition pattern of chromium in the respiratory tract is only partially understood. Although several studies have measured the accumulation of chromium in the lung or bronchus, none have assayed the distribution of the chromium accumulation in the lung or bronchus.9–11 A new device, the microscopic X-ray fluorescence analyzer with transmitted X-ray mapping imaging (XGT-2700; Horiba, Kyoto, Japan), was used in the current study to measure chromium accumulation in paraffin-embedded blocks.12

The goals of the current study were to understand the pattern of chromium deposition in bronchial and lung tissues and to evaluate the relation between premalignant changes in the bronchus and chromium accumulation in chromate workers.

MATERIALS AND METHODS

Tissue Specimens from the Bronchi or Lungs of Chromate-Exposed Workers with Lung Carcinoma

In a previous study, Hirose et al.13 evaluated microsatellite instability (MSI) in 38 lung tissue specimens from 31 chromate-exposed workers. In the current study, the distribution of chromium was measured in bronchial and lung tissue specimens from 10 of 31 chromate-exposed workers with lung carcinoma (Table 1). All patients were male. The mean age of the patients was 54.0 ± a standard deviation of 11.2 years (range, 40–74 years). The mean period of chromate exposure was 23.3 ± 8.4 years (range, 13–38 years). The mean Brinkman index (no. cigarettes per day × period [year] of smoking) was 382 ± 259 (range, 0–810), including 2 nonsmokers. Three patients had double lung carcinomas, and two patients had triple lung carcinomas. A previous study reported the replication error-positive (RER+) phenotype of these 10 lung carcinoma tissue specimens from chromate-exposed workers.13 The RER+ phenotype was defined as the presence of MSI at two or more loci, referring to the recommendation of the National Cancer Institute.14 Informed consent was obtained from all patients before sample collection, in accordance with institutional guidelines.

Table 1. Characteristics of Chromate Workers with Lung Carcinoma

Patient no.	RER status	Age (yrs)	Cr exposure (yrs)	Brinkman index	Multiple lung carcinoma	Cr accumulation (cps/mA)
RER: replication error; Cr: chromium; cps: counts per second; mA: mili ampere; ND: not detected.
1	−	56	14	450	Double	649.0
2	+	74	32	240		607.6
3	−	44	15	360	Triple	204.9
4	+	62	19	0	Double	172.2
5	+	46	26	810	Double	150.5
6	+	65	30	675		94.6
7	ND	40	13	390		61.1
8	+	47	21	520		14.7
9	−	45	25	370	Triple	9.7
10	−	61	38	0		4.4

Bronchoscopic Evaluation of Biopsy Specimens from Chromate-Exposed Workers

To detect lung carcinoma at an early stage, Yoshizawa et al.15 performed biopsies of the bronchus for 83 chromate industry workers in Tokushima, Japan, in 1982. These patients have been followed via sputum cytology evaluation every year until the present time. Biopsy under fiberoptic bronchoscopy was performed routinely at four locations (right bronchial spur between B¹ and B²⁺³, right bronchial spur between B⁷ and B⁸⁺⁹⁺¹⁰, left bronchial spur between B³ and B⁴, and left bronchial spur between B⁸ and B⁹). Biopsy was performed at the abnormal lesions if bronchoscopic examination found abnormal lesions. Two hundred seventy-three biopsy specimens of bronchus were obtained from 83 chromate workers with fiberoptic bronchoscopy. These biopsy specimens were stained with hematoxylin and eosin (H & E) and evaluated by two independent pathologists. There were no malignant lesions in these specimens. Histologic evaluation classified these specimens into six patterns: dysplasia, squamous metaplasia, goblet cell hyperplasia, basal cell hyperplasia, hypertrophy of basement membrane, and normal epithelia. If there were more than two abnormal patterns in one specimen, or if a worker had more than two abnormal patterns in four biopsy specimens, a more aggressive pattern was described as the diagnosis (dysplasia > squamous metaplasia > goblet cell hyperplasia > basal cell hyperplasia > hypertrophy of basement membrane > normal epithelia). Dysplasia was recorded in 3 specimens, squamous metaplasia in 14 specimens, goblet cell hyperplasia in 12 specimens, basal cell hyperplasia in 13 specimens, hypertrophy of basement membrane in 14 specimens, and normal epithelia in 27 specimens. We quantified the amount of chromium accumulation in the bronchi of 3 dysplasia specimens, 10 squamous metaplasia specimens, and 12 normal epithelia specimens, including 3 specimens from workers who were not involved in the manufacturing of chromate products (Table 2). All workers were male. Fourteen (56%) workers exhibited perforation of the nasal septum. The mean period of chromate exposure was 16.1 ± 8.4 years (range, 1–32 years). The mean Brinkman index was 325 ± 244 (range, 0–860), including 6 nonsmokers. Informed consent was obrained from all workers before sample collection.

Table 2. Characteristics of Chromate Workers Evaluated by Biopsy

Patient no.	Nasal perforation	Brinkman index	Cr exposure (yrs)	Histology	Cr accumulation (cps/mA)
+: with; −: without; Cr: chromium; cps: counts per second; mA: mili ampere; Sq-MP: squamous metaplasia; ep: epithelium.
39	+	625	25	Dysplasia	26.35
28	+	275	13	Dysplasia	17.76
4	+	860	28	Dysplasia	16.36
68	+	300	23	Sq-MP	46.46
56	+	400	23	Sq-MP	25.64
24	+	300	6	Sq-MP	25.55
51	+	0	15	Sq-MP	20.32
76	+	0	1	Sq-MP	18.62
15	+	720	8	Sq-MP	12.1
60	−	270	18	Sq-MP	11.77
19	+	680	25	Sq-MP	9.34
40	−	600	18	Sq-MP	8.2
74	−	0	8	Sq-MP	5.44
75	−	400	13	Normal ep	42.76
54	+	0	23	Normal ep	24.97
30	+	340	6	Normal ep	18.16
17	−	450	14	Normal ep	9.25
11	+	240	7	Normal ep	8.44
29	−	0	32	Normal ep	6.83
3	+	230	8	Normal ep	3.04
73	−	0	19	Normal ep	3.01
49	−	325	22	Normal ep	2.68
34	−	400	0	Normal ep	5.15
72	−	300	0	Normal ep	2.03
62	−	400	0	Normal ep	1.88

Measurement of Chromium Accumulation in Bronchial Tissue Specimens Using a Microscopic X-Ray Fluorescence Analyzer

We quantified the amount of chromium accumulation in the surgically resected tissue specimens or biopsy tissue specimens using a microscopic X-ray fluorescence analyzer with transmitted X-ray mapping imaging (Horiba).12 A fine X-ray beam was produced using a small X-ray tube and an X-ray guide tube (XGT) measuring 100 μm in diameter with a parabolic inner contour (Fig. 1A). The primary X-ray emitted from the X-ray tube is conducted by an XGT and irradiates the specimen. The fluorescent X-ray is induced from the sample when the primary X-ray irradiates the sample. The fluorescent X-ray is measured by an energy-dispersive silicon X-ray detector (Fig. 1B). The signal processed by the analyzer is further processed by a personal computer to obtain qualitative and quantitative information. This instrument can simultaneously analyze 13 elements, from sodium to uranium. An image of the distribution of elements delivered from the fluorescent X-ray is obtained when the sample stage is moved in the x and y directions by motors. We can obtain the image of X-rays transmitted through the sample using the detector, which is located at the bottom of the sample. Scanning images of chromium accumulation were produced. The intensity of chromium levels was measured at the area of maximum intensity and at two or three other randomly selected areas. When a standard material with a chromium content of 114.6 mg/kg polyethylene was analyzed using this device, the chromium content was determined to be 6.71 counts per second (cps)/mili ampere (mA).

Figure 1. The microscopic X-ray fluorescence analyzer with transmitted X-ray mapping imaging. (A) An X-ray guide tube (XGT) measuring 100 μm in diameter. (B) Construction of the microscopic X-ray fluorescence analyzer. Pa: pascals; WD: width.

To evaluate the relation between chromium accumulation and histopathologic fingings, we combined the scan images of chromium (red) and sulfur (green) accumulation. Because the intensity of the primary X-ray and the measurement time are constant, the quantity of fluorescent X-ray radiation increases in a small analytic area compared with a large area. In the current study, imaging of chromium accumulation (red spot) in biospy samples is more sensitive than in resected specimens. Each formalin-fixed and paraffin-embedded block was sectioned at 3 μm and stained with H & E. Two independent observers (K.K. and Y.T.) evaluated the histopathologic findings in the loci of H & E-stained sections, which corresponded to chromium accumulation (red spots). In the resected lung tissue specimens, 24 loci of chromium accumulation (red spots) were evaluated.

Statistical Analysis

Statistical analysis involved the unpaired or paired t test, the Pearson correlation test, and the Spearman correlation coefficient using SPSS for Windows (Version 11.0.1; SPSS, San Diego, CA). Significance was indicated by a P value less than 0.05.

RESULTS

Amount of Chromium Accumulation in Surgically Resected Lung or Bronchial Tissue Specimens

The accumulation of chromium in 10 resected lung tissue specimens, including 1 lobar bronchus and 3 segmental bronchi, was measured with the XGT-2700. Figures 2 and 3 show representative specimens of the proximal bronchus and the peripheral lung. The tissue specimen in Figure 2 was obtained from Patient 6 who had an early squamous cell carcinoma of the left bronchus between the main bronchus and lower lobe bronchus. He underwent a sleeve resection of the left lower lobe. The section showed the bifurcation of the basal bronchus and the B⁶ bronchus. Figure 2A shows the scanning image of chromium (red) and sulfur (green) accumulation and Figure 2B shows the corresponding H & E staining image using a low-power field. There was a large accumulation of chromium in the lymph node with anthoracotic lesions around the basal bronchus (Fig. 2C). Figure 2D shows the histogram of intensity of various elements at the point of maximum intensity of chromium. The intensity of chromium was 94.6 cps/mA.

Figure 2. Bifurcation of the left basal bronchus and left B⁶ bronchus in Patient 6. (A) Scanning image of chromium (red) and sulfur (green) accumulation using the microscopic X-ray fluorescence analyzer. (B) The corresponding hematoxylin and eosin (H & E)-stained image in a low-power field. (C) Histopathology of a spot exhibiting chromium accumulation (rectangular region in B; H & E, original magnification ×40). (D) Histogram of intensity of each element at the point of maximum chromium intensity. VFS: virtual full scale; cps: counts per second; keV: kilo-electron volts.

Figure 3. Left upper lobe of Patient 2. (A) Scanning image of chromium (red) and sulfur (green) accumulation using the microscopic X-ray fluorescence analyzer. (B) The corresponding hematoxylin and eosin (H & E)-stained image in a low-power field. (C) Histopathology of a spot exhibiting chromium accumulation (square region 1 in B): subpleural region with anthoracotic lesions. (D) Histopathology of a spot exhibiting chromium accumulation (square region 2 in B): bronchiole with anthoracotic lesions. (C,D: H & E staining, original magnification ×40.)

Figure 3 shows the tissue specimen obtained from Patient 2, who had an advanced squamous cell carcinoma of the left upper lobe. He underwent a left upper lobectomy. A large accumulation of chromium was observed in the subpleural regions and lung parenchyma (Fig. 3A,B). Microscopic evaluation of the subpleural region with chromium accumulation (270.6 cps/mA) showed slightly thickened pleura with anthoracotic lesions (Fig. 3C). In addition, on microscopic examination, the region with the greatest amount of accumulation (607.6 cps/mA) exhibited a bronchiole with anthoracotic lesions (Fig. 3D).

Chromium Accumulation and Clinical Findings in Chromate-Exposed Workers with Lung Carcinoma

The mean chromium accumulation in 10 specimens was 197 ± 238 cps/mA (range, 4–649 cps/mA; Table 1). There was no chromium accumulation (red spot) in the measured areas of three resected lung specimens. The maximum chromium accumulation levels in these 3 specimens ranged from 4.4 to 14.7 cps/mA. The pattern of chromium accumulation was diffuse only in the lung tissue specimen of Patient 2 (Fig. 3). The maximum accumulation was 608 cps/mA. In the other six specimens, the pattern of chromium accumulation was scattered. The maximum accumulation ranged from 61 to 649 cps/mA. There was no chromium accumulation (red spots) in the lobar or segmental bronchi of four resected lung tissue specimens (Fig. 2). Anthoracotic lesions were recognized microscopically in 21 (87.5%) of 24 peripheral loci (bronchioles and subpleural regions) with chromium accumulation (red spots), as shown in Figure 3. There was no correlations between the amount of chromium accumulation and any clinical findings (period of chromate exposure, presence of multiple lung carcinoma, RER status, Brinkman index and age; Table 1).

Amount of Chromium Accumulation in Bronchial Tissue Specimens Obtained by Bronchoscopy

The XGT-2700 was used to measure chromium accumulation in 90 biopsy specimens from 25 workers. Figure 4 shows a representative tissue specimen, which was obtained from Patient 68, who had a squamous metaplasia of the right spur between the B¹ and B²⁺ bronchi. Figure 4A shows the map of chromium (red) and sulfur (green) accumulation, and Figure 4B shows the corresponding H & E staining image in a low-power field. There was a spot exhibiting a large amount of accumulation in the subepithelial region of the bronchus (Fig. 4A,B). Figure 4C shows the histogram of intensity of various elements at this spot. The intensity of chromium accumulation was 46.46 cps/mA.

Figure 4. Biopsy specimen from right bronchial spur between B¹ and B²⁺³ in Patient 68. (A) Scanning image of chromium (red) and sulfur (green) accumulation using the microscopic X-ray fluorescence analyzer. (B) The corresponding hematoxylin and eosin (H & E)-stained image in a low-power field (H & E staining, original magnification ×40). (C) Histogram of intensity of each element at the point of maximum chromium intensity. VFS: virtual full scale; cps: counts per second; keV: kilo-electron volts.

Chromium Accumulation and Clinical Findings in Chromate-Exposed Workers

We measured the chromium accumulation in 90 biopsy specimens. Chromium was detectable in 63 samples (70%). The mean accumulation was 6.5 ± 9.2 cps/mA (range, 0–46.5 cps/mA). In biopsy specimens obtained from 19 workers for whom we could make measurements at 4 locations of the bronchi, we evaluated the correlation between chromium accumulation and the location of the bronchi (right upper lobe [RUL], right lower lobe [RLL], left upper lobe [LUL], and left lower lobe [LLL]). The mean chromium accumulations in the RUL, the RLL, the LUL, and the LLL were 4.60 ± 11.03, 5.07 ± 5.57, 7.54 ± 8.92, and 9.21 ± 12.19, respectively. There was a significant difference in chromium accumulation between bronchi of the RLL and bronchi of the LLL (5.07 ± 5.57 vs. 9.21 ± 12.19; paired t test: P = 0.021).

The maximum amount of chromium accumulation for each worker is shown in Table 2. Figure 5 shows the correlation between histologic change and chromium accumulation in biopsy specimens. The mean chromium accumulation in chromate workers with dysplasia (n = 3) was 20.2 ± 5.4 cps/mA (range, 26.4–16.4 cps/mA). The mean levels of chromium accumulation in chromate workers with squamous metaplasia (n = 10), chromate workers with normal bronchial epithelia (n = 9), and non-chromate workers with normal bronchial epithelia (n = 3) were 18.3 ± 12.2, 13.2 ± 13.4, and 3.0 ± 1.8 cps/mA, respectively. The amount of chromium accumulation significantly increased according to the progression of malignant change in the bronchial epithelium (normal epithelium → squamous metaplasia → dysplasia; Spearman correlation coefficient: r = 0.576; P = 0.003).

Figure 5. Correlation between the histology of the biopsy specimen and the maximum level of chromium accumulation in chromate workers. P = 0.003 (Spearman rank test). cps: counts per second; mA: mili ampere.

Figure 6 shows the correlation between the period of chromate exposure and chromium accumulation. There was no correlation between them (Pearson correlation test: r = 0.262). The chromium accumulation of workers with perforation of the nasal septum (19.5 ± 10.5) was significantly higher than that of workers without perforation (9.0 ± 11.6; P = 0.03).

Figure 6. Correlation between the period of chromate exposure and the level of chromium accumulation. r = 0.262 (Pearson correlation test). cps: counts per second; mA: mili ampere.

DISCUSSION

In the 1980s, several studies used atomic absorption spectrometry (AAS) to measure the chromium content in the lungs of chromate workers with lung carcinoma.9, 10 AAS requires 2–5 g of tissue for 1 measurement. This method can measure the chromium content of the whole sample, but it cannot be used to determine the histologic distribution. Ishikawa et al.11 reported a new method of measuring chromium content using neutron irradiation. They measured the levels of chromium from the trachea to subsegmental bronchi and demonstrated that chromium concentrations were higher at airway bifurcations than elsewhere. However, there have been no reports on chromium accumulation in the bronchi of more than subsubsegmental bronchus or alveolar regions. In the current study, we used the XGT-2700 to measure the accumulated levels of chromium in bronchial and lung tissue specimens.12 This new method can be used to measure the levels of chromium in situ. Because the sample on the stage is moved along the x and y axes under primary X-ray irradiation, fluorescent X-rays are measured at every point. The XGT-2700 draws an image of the chromium accumulation in all fields of the sample and expresses the intensity of chromium accumulation in a histogram at each point. Using this device, we could microscopically evaluate the chromium accumulation in the bronchial tree and alveolar regions.

The current study reported that there were regions with high levels of chromium accumulation (red spots) in some bronchioles and subpleural regions, but not in the lobar or segmental bronchi. The red spots appeared in tissue specimens with maximum chromium accumulations of more than 61.1 cps/mA. We believe that the level of chromium accumulation in the lobar or segmental bronchi is less than 60 cps/mA. Chromium accumulation ranging from 0 to 46.5 cps/mA was detected in the stroma of the bronchi (first or second generation) of biopsy specimens. These data demonstrated that chromium accumulation in the proximal bronchi (lobar or segmental bronchus) was lower than in the bronchioles and subpleural regions in the lung. Sano6 reported that most of the chromate particles were 1–3 μmin diameter and that their shape was approximately spherical according to microscopic measurements of lung tissue specimens obtained at autopsy. Lippmann et al.16 demonstrated that the deposition of particles 1–3 μm in diameter in the airway generated 2 peaks. One peak (3–5%) represented the 3rd or 4th generation (the subsegment or subsubsegment bronchus in the tracheobronchial tree), and the other (9–15%) represented the 22nd generation (alveolar region). Considering the size of chromium particles, the finding in the current study that chromium accumulation was greater in the bronchiole and in the alveolar regions than in the proximal airways was reasonable.

Most of the spots exhibiting high levels of accumulation (red spots) in the bronchioles and subpleural regions coincided with the anthoracotic lesions of the lung. Kim et al.17 also reported similar findings using an X-ray microanalyzer. Schulz et al.18 demonstrated that the deposition rate of 0.01–0.1 μm particles in the alveolar region is the same as that of 1–4 μm particles. Given that the size of tobacco smoke particles ranges from 0.01 to 0.4 μm, the deposition of chromium particles (1–3 μm) showed a similar distribution to that of tobacco smoke particles in the peripheral region of the lung.

The current study on the finer topographic determination of chromium showed that the chromium particles detected in the bronchial tissue specimens were deposited in the bronchial stroma, but not in the epithelium of tissue specimens. Bronchial clearance is essentially completed within the first postexposure day.16 Ciliary clearance removed particles from deposition sites along the tracheobronchial tree to the pharynx, from which they were removed to the gut by swallowing. Ninety percent or more of the initial deposit is removed from the bronchial tree through the bronchial duct.16 We believe that some chromium particles remain in the stroma of the bronchi and the interstitium of the lung or subpleural regions and that some phagocytosed chromium particles remain in the lymph nodes.

The current study showed that the accumulation of chromium in the resected lung tissue specimens or biopsy specimens in chromate workers was not associated with the period of chromate exposure. Previous studies also showed that the level of chromium in the resected lung tissue specimens was not correlated with the duration of chromate exposure in chromate workers with lung carcinoma.19–22 Two reasons for this findings are considered. First, the degree of exposure to chromate varies according to the stage of the process involved in making chromate products.22 Because chromate workers are switched from one process to another several times throughout their careers, it is difficult to ascertain the extent to which chromate exposure is a direct result of working in the chromate industry. Second, the retention of inhaled particles depends on several factors, including particle size, shape, solubility, and surface chemistry.23 Chromic acid and sodium dichromate are highly soluble, whereas strontium, calcium, and zinc chromate are moderately soluble. However, lead and barium chromate are insoluble.24 It is possible that the solubility of the chromate affects the retention of the inhaled particles.

The current study reported that the amount of chromium accumulation significantly increased according to the progression of malignant change of the bronchial epithelium (normal epithelium → squamous metaplasia → dysplasia). Auerbach et al.25 reported that the frequencies of CIS in nonsmokers, smokers consuming less than 1 pack of cigarettes per day, and smokers consuming more than 1 pack per day were 1%, 4.1%, and 6%, respectively. Many of the prospective studies provided evidence that a gradient of increasing risk for lung carcinoma mortality is parallel to increasing numbers of cigarettes smoked per day.26 These data indirectly demonstrate that the progression of malignant change depends on the amount of carcinogen in tobacco smoke-induced lung carcinoma. Our results showed the same correlation between the development of malignant change and chromium accumulation in the stoma of the bronchi. Thus, we hypothesize that certain genetic alterations become more intensive in proportion to the amount of chromium accumulation in bronchial stoma. Consequently, bronchial lesions of various histologic grades (squamous metaplasia, dysplasia, CIS) are induced.

Previous studies reported that chromate-induced lung carcinomas infrequently exhibited p53 gene mutations, which are common in non-chromate-induced lung carcinoma, and that activation of Ki-ras and Ha-ras genes due to point mutations in chromate-induced lung carcinoma is a rare event.27, 28 There was no significant difference in loss of heterozygosity (LOH) at 3p between chromate-induced lung carcinomas and non-chromate-induced lung carcinomas.13 In contrast, the frequency of RER in chromate-induced lung carcinomas (78.9%) was significantly higher than in non-chromate-induced lung carcinoma (15.4%).13 Sozzi et al.29 reported that MSI was found not only in lung carcinoma (32%) but also in normal epithelia (36%). Wistuba et al.30 reported a progressive increase in LOH frequency according to increasing severity of histopathologic changes in squamous cell carcinoma. Others have also pointed out that the incidence of aberrant promoter methylation of the p16 tumor suppressor gene gradually increased with malignant progression in preneoplastic lesions of human squamous cell carcinoma.31, 32 A previous study performed by our group demonstrated that 9 of 10 early squamous cell carcinomas of the proximal bronchi exhibited MSI in chromate workers with lung carcinoma.13 Currently, we are evaluating MSI, LOH, and methylation of the p16 gene in these specimens (an early malignancy, dysplasia, squamous metaplasia, and normal epithelia) with chromate exposure. Clarification of the correlation between genetic aberrations and chromium accumulation in these specimens will aid in elucidating the mechanism of carcinogenesis in chromium-induced lung carcinoma.