Environ. Sci. Technol. 2006, 40, 6176-6180
Induced CYP1A2 Activity as a Phenotypic Biomarker in Humans Highly Exposed to Certain PCBs/PCDFs GEORGE H. LAMBERT,† LARRY L. NEEDHAM,‡ WAYMAN TURNER,‡ TE JEN LAI,§ DONALD G. PATTERSON, JR.,‡ AND Y . L E O N G U O * ,⊥ Center for Child and Reproductive Environmental Health, Department of Pediatrics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, NJ, USA, and Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA, and Department of Psychiatry, Chung Shan Medical University Hospital, Taichung, Taiwan, and Department of Environmental and Occupational Medicine, College of Medicine, National Taiwan University and National Taiwan University Hospital, Taipei, Taiwan
Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) continue to be a worldwide public health concern due to their levels in the environment and humans, and associated adverse health effects. In animals, one of the most sensitive effects of physiologically significant body burdens has been the induction of cytochrome P450 1 (CYP1) family of enzymes. This study examined the capacity of CYP1 enzyme induction to be a biomarker of exposure to a mixture of PCBs and PCDFs and of adverse human health effects. We followed a group of people highly exposed to PCBs and PCDFs due to accidental ingestion of contaminated rice oil, the Yucheng cohort. A total of 174 Yucheng and 134 control subjects were studied. The caffeine breath test, a monitor of CYP1A2 activity, was conducted, and its results were compared to serum levels of chemicals and the subjects’ medical history. Total dioxin serum toxic equivalency (TEQ) in the Yucheng cohort and their controls were 577 ( 393 ppt lipid and 21 ppt lipid, respectively. CYP1A2 activity was elevated in Yucheng subjects more than 2-fold and correlated with serum TEQ (R2) 0.62). Manifestations like chloracne, fingernail abnormalities, and headaches were well predicted by P4501A2 activity. It is concluded that CYP1A2 induction seen in the Yucheng cohort is an excellent biomarker of exposure and human health effects in individual subjects and cohort.
Introduction Polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs), and related compounds are among the most * Corresponding author phone: +886-2-33228215; fax: +886-23322-8216; e-mail:
[email protected]. † University of Medicine and Dentistry of New Jersey. ‡ Center for Disease Control and Prevention. § Chung Shan Medical University Hospital. ⊥ National Taiwan University and National Taiwan University Hospital. 6176
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 19, 2006
widespread environmental pollutants (1); PCBs have been routinely detected in wildlife as well as human tissue samples since the 1960s. PCDFs and PCDDs became more noticeable in recent years, after more reliable analytical methodologies were developed for these compounds. Multiple adverse health outcomes including those of the skin, liver, neurological, and reproductive systems have been reported in humans and animals. Due to the long half-lives of these chemicals in the environment and in animals and humans, they will be a concern for many years to come (2). The toxic capacity of these chemicals to humans have been a worldwide medical, public health, and legal concern resulting in hundreds of millions of dollars in the clean up of these chemicals. PCDDs and PCDFs exert their toxic effects through binding to the aryl hydrocarbon receptor (AhR). Coplanar PCBs also bind to AhR with smaller efficacy, and thus are considered to possess dioxin-like toxic effects. Through AhR binding, one of the most important and well-documented effects is induction of the cytochrome P-450 (CYP) superfamily of isoenzymes in animals, especially CYP 1A1 and 1A2. In animal studies, health effects caused by dioxin-like chemicals have been shown to correlate with the capacity of the congener mix to induce the cytochrome P450 family I enzymes through interaction with the Ah receptor. In addition, CYP1A2 is involved in the metabolic activation of several carcinogens such as mycotoxins, aromatic and heterocyclic amines, and nitrosamines (3). Therefore, the induction of CYP1A2 activity has been considered valuable biomarkers for exposure of PCDDs, PCDFs, and dioxin-like PCBs. CYP1A2 has been detected primarily in the liver in humans and cannot be assayed from peripheral blood samples. Therefore, direct measurement of CYP1A2 activity has been less possible in epidemiological studies. The activity of CYP1A2 can be measured by using the production rate of caffeine metabolites through 3-N-demethylation, which is catalyzed by CYP1A2. Such an approach has been utilized in the survey of exposure to PCBs and dioxin-like chemicals (4). However, it is important for a biomarker to be predictive of health outcomes, as well as be related to exposure. In order to address the question of whether P450 1A2 induction is a biomarker of exposure to some of PCBs and dioxin-like chemicals, and if the induction correlates to adverse health effects, the caffeine breath test (CBT), a measure of cytochrome P4501A2 activity, was conducted in a cohort of people highly exposed to PCBs and PCDFs in central Taiwan, and compared to the subjects serum levels of PCBs and PCDFs, as well as with disease outcomes in the exposed individuals. In 1978-1979, a mass poisoning occurred in central Taiwan from rice oil contaminated by heat-degraded PCBs (5). Symptoms included chloracne, hyperpigmentation, and peripheral neuropathy, and the illness was referred to as “Yucheng” (oil disease in Chinese). By 1983, a registry set up and maintained by the Taiwan Provincial Department of Health included 2061 subjects (6). Patients used the contaminated cooking oil for an average of 8 months; they were estimated to have consumed about 1 gram of PCBs and 3.8 mg of PCDFs (7) (mostly the 2,3,4,7,8-penta-CDF and the 1,2,3,4,7,8-hexa-CDF) during that time, resulting in median serum PCB levels of 60 ( 39 parts per billion wet weight, and serum total PCDF levels of 140 ( 70 parts per trillion (ppt) wet weight (8), or approximately 36 000 ( 18 000 ppt lipid, assuming 0.4% serum lipid in this population. Some of these chemicals are extremely persistent in human tissue; 14 years after exposure, PCB and PCDF measurement in 56 women from the cohort showed that total PCBs were still 7-fold higher and PCDF congeners 40-130 times higher than that of pooled 10.1021/es0608646 CCC: $33.50
2006 American Chemical Society Published on Web 08/25/2006
local controls (9). We tested the hypotheses that CYP1A2 activity was related (a) to serum levels of PCBs and PCDFs as summarized by TCDD toxic equivalencies; and (b) to clinical manifestations caused by PCBs/PCDFs intoxication.
Materials and Methods The study protocol was approved by the Institution Review Board at National Cheng Kung University Medical College and University of Medicine and Dentistry of New Jersey (UMDNJ)sRobert Wood Johnson Medical School, and complied with the principles outlined in the Declaration of Helsinki (10). All subjects signed informed consent before enrolling in the study. In 1993, those Yucheng subjects and their matched controls aged 30 years or older were interviewed by phone (6) for complete review of medical history and clinical manifestations. Information was obtained from 795 exposed and 693 control subjects. Lifetime prevalence of chloracne, abnormal nails, hyperkeratosis, skin allergy, goiter, headache, gum pigmentation, and broken teeth were found more frequently in the exposed men and women. These findings were our clinical outcomes to be compared with CYP1A2 activity. For CYP1A2 activity measurements, the caffeine breath test was conducted. Potential adult volunteers received an introductory letter that outlined the purpose, procedures, and risks of the study. All selected subjects were subsequently visited by a field epidemiologist who discussed the study and obtained a verbal commitment to participate in the study. One week prior to the study day, a letter was sent to the subjects to remind them of the appointment day and protocol requirements. Volunteers were asked to refrain from ingesting products which contained methyl xanthines, such as chocolate or caffeine, for 24 h prior to the clinic visit and not to eat or drink anything except water after midnight prior to the clinic visits, which was scheduled between 7:30 and 9:30 A.M. Volunteers whose medical history met the protocol criteria and whose physical examination and urinalysis were normal in the morning of the study were entered into the caffeine study. Informed consents were obtained from the participants before the test. The methodology of caffeine breath test was detailed elsewhere (11). Because 3-N-demethylation of caffeine is catalyzed by CYP1A2, caffeine metabolism reflects hepatic CYP1A2 activity (12). The caffeine substrate used to monitor caffeine 3-N-demethylation in the CBT was [3-13C-methyl] caffeine (99% 13C) synthesized by Cambridge Isotope (Cambridge, MA). The labeled caffeine dose, 3 mg/kg (up to a maximal dose of 200 mg), was dissolved in 20 mL of sterile water and ingested by the volunteers, followed by ingestion of a 20-mL water wash of the container. All subjects sat quietly for 15 min before and throughout the CBT. A breath sample was obtained by having the participants blow into a plastic bag just prior to and after the ingestion of the labeled caffeine at 30 and 60 min. Twenty milliliters of the expired air was removed from the bag by a syringe and injected into a nonsterile, plain, nonsilicone-coated Venoject tube (Terumo Medical Corp., Elkton, MD) for transport and storage. The 13CO /12CO ratio was determined by differential gas-isotope 2 2 ratio mass spectrometry (13). The excess 13C is calculated from the ratio found in the breath sample just before and after ingestion of the 13C-labeled caffeine and the labeled CO2 exhaled was calculated as percent labeled dose exhaled per hour (14). For the measurements of the serum levels of PCB, PCDF, and PCDD congeners, the archived serum samples, drawn in 1994-1995, stored at -80 °C, were sent on dry ice to the U.S. Centers for Disease Control and Prevention. Highresolution gas chromatography/mass spectrometry (15) was used for the measurement of 2,3,7,8-tetrachloro-dibenzodioxin (TCDD), 1,2,3,7,8-pentachlorodibenzodioxin (PnCDD),
1,2,3,4,7,8-hexachlorodibenzodioxin (HxCDD), 1,2,3,6,7,8HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-heptachlorodibenzodioxin (HpCDD), 1,2,3,4,6,7,9-HpCDD, octachlorodibenzodioxin (OCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-PnCDF,2,3,4,7,8-PnCDF,1,2,3,4,7,8-HxCDF,1,2,3,6,7,8HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8HpCDF, 1,2,3,4,7,8,9-HpCDF, OCDF, 3,3,4,4-TCB (IUPAC 77), 3,4,4′,5-TCB (IUPAC 81), 3,3′,4,4′,5-PnCB (IUPAC 126), and 3,3′,4,4′,5,5′-HxCB (IUPAC 169). Ortho-substituted PCBs were analyzed by a Hewlett-Packard 5890 gas chromatograph (Hewlett-Packard, Houston, TX) using an electron-capture detector, including 2,3,3′,4,4′-PnCB (IUPOAC 105), 2,3′,4,4′,5PnCB(IUPAC118),2,3,3′,4,4′,5-HxCB(IUPAC156),2,3,3′,4,4′,5′HxCB (IUPAC 157), 2,3′,4,4′,5,5′-HxCB (IUPAC 167), and 2,3,3′,4,4′,5,5′-HpCB (IUPAC 189). Values were reported on a lipid weight basis in parts per trillion (ppt) by dividing the congeners on a whole-weight basis by total serum lipid content, estimated from measurements of triglycerides, and total cholesterol (16). TCDD toxic equivalency (TEQ) was calculated as the sum of the product of each analyte concentration and its TEF (17). The median serum sample weight for these samples was 1.35 g, and the median lipid-adjusted limits of detection were 15.8 ppt for 2,3,4,7,8-pentaCDF (ranging 8.1 ppt to 37.2 ppt) and 6.7 ppt for 1,2,3,4,7,8hexaCDF (ranging 2.7 ppt to 25.1 ppt). For those 56 serum samples of Yucheng individuals collected in 1994, estimates of TEQ were calculated by using the half-life of 7.7 years (18) to compare with CBT in 1995. The data were entered to the JMP statistical package (SAS Company) for analysis. The CBT parameter was compared between exposed and unexposed individuals by Wilcoxon rank-sum test. The CBT parameter was compared with serum levels of TEQ using regression analysis. The relationship between the clinical presentations and CBT in 60 min was analyzed by logistic regression analysis.
Results A caffeine breath test was performed on 308 subjects (174 exposed and 134 controls) in 1995. Age, education, and smoking history in Yucheng and control subjects at time of the caffeine breath test are shown in Table 1. Serum levels were successfully measured among 36 Yucheng men and 56 Yucheng women (Table 2). Despite the relatively small amount of serum samples approved by the steering committee, the most important congeners 2,3,4,7,8-PnCDF (median detection limit 15.8 ppt; ranged 8.1 ppt to 37.2 ppt) and 1,2,3,4,7,8-HxCDF (median detection limit 6.7 ppt; ranged 2.7 ppt to 25.1 ppt) were detectable in more than 95% of Yucheng individuals who donated blood for this study. For exposure information among control individuals, background levels of PCDDs and PCDFs were measured to be 15.2 ng TEQ/kg lipid for men and 17.0 ng TEQ/kg lipid for women (19). Background levels of PCBs was available only in lipidadjusted milk samples in women (20). Background levels of TEQ were thus estimated by summarizing dioxin, furan TEQ, and the women’s milk PCB TEQ. The CBT parameter used for comparison was % 13Clabeled exhaled over 1 h and was compared by gender groups, smoking status, and age in exposed and unexposed subjects (Table 3). Yucheng subjects had much higher CBT parameter than the controls. This was not changed by stratifying subjects by sex or smoking status. In unexposed people, smokers had higher rate of CYP1A2 activity. Regression analysis was done between CBT parameter and serum levels of TCDD TEQ. CBT parameter is highly associated with serum TEQ (Figure 1). The relationship between reported clinical manifestations and CBT parameters were compared by logistic regression. Among Yucheng subjects, the CBT parameter was categorized into 4% (N ) 33). Risks VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Age, Education, and Smoking History of Yucheng and Control Subjects, at time of Caffeine Breath Test, 1995, Taiwan men
subject No. Age, year Distribution 30-39 (%) 40-49 (%) 50-59 (%) 60-69 (%) >70 (%) education, yr currently smoking
women
yucheng
reference
p-value
yucheng
reference
P value
71 53.3 ( 12.6
48 54.3 ( 13.1
n.s.
103 45.3 ( 11.9
86 43.8 ( 11.3
n.s.
19.7 22.5 21.1 28.2 8.5 7.3 ( 3.6 42.2%
25.0 8.3 35.4 16.7 14.6 7.7 ( 3.7 54.2%
52.4 19.4 10.7 14.6 2.9 6.4 ( 4.0 1.0%
58.1 17.4 12.8 8.1 3.5 6.4 ( 4.7 2.3%
n.s. n.s. n.s.
n.s. n.s. n.s.
TABLE 2. Serum Levels (mean ( standard deviation) of PCDDs, PCDFs, and PCBs Toxic Equivalencies (ppt TEQ, lipid) in Yucheng Men and Women in 1994-1995, as Compared to Background Reference, Taiwana men
subject no. dioxin TEQ furan TEQ PCB TEQ total TEQ
women
yucheng
reference
yucheng
reference
36 3.6 ( 11.0 315.1 ( 284.5 133.4 ( 97.6 452.1 + 317.8
8.0 ( 6.2b 7.3 ( 5.3b not available 20.1d
56 7.1 ( 9.7 527.5 ( 357.1 127.0 ( 91.4 657.6 + 417.9
9.0 ( 5.5b 8.0 ( 5.4b 4.9 ( 9.4c 21.9d
a Average age and years of education were compared by unpaired T-test; age distribution and smoking history were compared by Chi-square test. b Reference levels from Chen et al. (ref 19), furan/dioxin TEQ was estimated by summarizing 2,3,7,8-TeCDF, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8,9-HpCDF, Ocdf, 2,3,7,8-TeCDD, 1,2,3,7,8PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, and OCDD TEF values. c Referece levels of lipid-adjusted milk PCB level from Chao et al. (ref 20), PCB TEQ was estimated by summarizing TEF values of PCB congeners IUPAC 77, 81, 126, 169, 105, 114, 118, 123, 156, 157, 167, and 189. d Estimated by summarizing background dioxin and furan TEQ, and the PCB TEQ in women’s milk.
TABLE 3. CYP1A2 Activity as Determined by Cumulative Exhalation of Labeled C13 in 1 h by Exposure, Gender, and Smoking Status. Tests were Done Using Wilcoxon Rank-sum Testa gender currently smoking
male female no yes
age male nonsmokers male smokers female nonsmokers female smokers
N
exposed
N
unexposed
P-value
71 103 143 31
2.46 ( 0.14 3.20 ( 0.12 2.96 ( 0.14 2.88 ( 0.30 nonsignificant 2.30 ( 0.20 2.86 ( 0.25 3.20 ( 0.12 3.49
48 86 106 28
1.30 ( 0.17 1.12 ( 0.13 1.17 ( 0.07 1.46 ( 0.12b nonsignificant 1.13 ( 0.29 1.48 ( 0.28 1.12 ( 0.13 1.29 ( 0.63
P < 0.001 P < 0.001 P < 0.001 P < 0.001
41 30 102 1
22 26 84 2
a There were only one female smoker in exposed group and two in the control, thus a comparison was not made. non-smokers.
b
P)0.0011 P < 0.001 P < 0.001 Not tested P < 0.05 compared with
TABLE 4. CBT Parameter as a Predictor of Risk of Having Clinical Manifestations in Yucheng Patientsa CBT
abnormal nail
skin allergy
chloracne
hyperkeratosis
gum pigmentation
headache
4%
1 2.04 (0.66-6.10) 3.90* (1.37-11.3)
1 0.68 (0.26-1.66) 1.11 (0.44-2.68)
1 1.43 (0.52-3.76) 3.09* (1.21-7.90)
1 2.45 (0.65-8.92) 2.84 (0.75-10.5)
1 1.42 (0.27-6.25) 3.63 (0.99-13.9)
1 0.93 (0.14-3.68) 3.84* (1.23-11.10)
a Entities are odds ratios (95% confidence intervals) of developing each clinical manifestation using lowest CBT parameter (
