Mar 5, 1995 - before the concentrate was free of visible black oil. The refined ... Hansen 1994; Safe 1994); nevertheless, a useful ranking is achieved and relative ..... exposed to a thousand-fold lower dose of TCDD in utero (Seo et al. 1995) ...
Arch. Environ. Contam. Toxicol. 29, 334-343 (1995)
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OF
Environmental Contamination a n d Toxicology © 1995Springer.VerlagNew York Inc.
Environmental Polychiorinated Biphenyls: Acute Toxicity of Landfill Soil Extract to Female Prepubertal Rats L. G. Hansen, 1 M.-H. Li, 1 A. Saeed, 1 B. Bush 2 1 Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Avenue, Urbana, Illinois 61801, USA 2 Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, New York 12201, USA Received: 26 November 1994/Revised: 5 March 1995
Abstract. Subsurface soil from a National Priorities List landfill containing about 2.5% polychlorinated biphenyls (PCBs) was extracted and the extract cleaned by Florisil ® slurry and alumina column chromatography. The refined extract contained 48 mg/mL PCB, mainly trichlorobiphenyls and tetrachlorobiphenyls, traces of polychlorinated naphthalenes, 125 txg/mL 2,2-bis-p-chlorophenyl- 1,1-dichloroethylene (DDE), and low levels of chlorinated dibenzofurans. The refined extract was dissolved in corn oil and administered intraperitoneally to weanling (day 20) female rats on days 20 and 21; rats were terminated on day 22. Limited data indicated possible hematopoietic effects, including neutrophilia. There were no changes in relative uterus, kidney, or adrenal gland weights between total doses of 3 to 96 mg/kg total PCB. Relative liver weights increased significantly at 36 mg/kg and activities of P450s 1A1 (as ethoxyresorufin O-dealkylase) and 2B (as pentoxyresorufin O-dealkylase) increased at 12 mg/kg and plateaued at 36 (P450 1A1) or 48 (P450 2B) mg/kg. Serum total thyroxine (T4) declined significantly at doses of 36 mg/kg and greater; thyroid follicular epithelial cells were significantly larger within the same dose range, but the follicular colloid area decreased to less than 60% control values at 12 mg/kg and remained at this size through 72 mg/kg. Maximum mobilization of T4 apparently occurred at 12 mg/kg and attenuated measured declines in circulating levels, Even though a large proportion of proven and probable estrogenic chlorobiphenyls (CBs) were present, the lower amounts of more potent antiestrogenic aryl hydrocarbon (Ah) receptor agonists and/or decreased responsiveness because of low serum T4 levels may have antagonized the uterotropic response.
Polychlorinated biphenyls (PCBs) are widespread environmental contaminants with a broad range of biological activities (Safe et al. 1987; Safe 1990; Hansen 1987, 1994). Although worldwide commercial production declined dramatically in the
Correspondence to: L. G. Hansen
1970s and has essentially ceased, used electrical equipment and leaking disposal sites are current sources of environmental hot spots. Fish and seafood from the more contaminated areas are dietary sources of major concern (Safe et al. 1987; Hansen 1987), but pulses of dietary exposure may occur due to contamination of produce from unexpected sources (e.g., Hansen et al. 1989). More consistent background exposure can occur from atmospheric sources (Hornbuckle et al. 1992). In addition to dispersion of waste PCBs from hot spots, minor modern sources include chlorination of waste water (Voudrias et al. 1986) and crosslinking of silicone rubber (Perdih and Jan 1994). The net effect of environmental PCBs depends upon interactions of both chlorobiphenyls (CBs) and biological systems as well as the individual actions of specific congeners so that accurate risk assessment is difficult. It is important to determine multiple toxic actions of different mixtures of CBs from different environmental sources so that proposed predictive indices can be compared to real biological data. Currently, the most accepted index for PCB toxicity is the Toxic Equivalency Factor (TEF) (Safe 1990; Ahlborg et al. 1994). One of the earliest and most often measured responses to PCBs is induction of hepatic cytochromes P450; the specific pattern of induction can be predictive of the types of effects expected (Safe 1990). The most potent PCBs are those with lateral chlorines and less than 2 ortho chlorines; these congeners can assume a planar conformation by stacking interactions with receptors and produce dioxin-like effects, including induction of CYP1A1 and hormone disruption (McKinney and Waller, 1994). The potencies of mixtures and environmental samples for causing these effects can readily be estimated by comparing key actions to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Safe 1990, 1994; Ahlborg et al. 1994). The most responsive parameter is induction of the CYP1A1 gene, most often determined by measuring ethoxyresorufin-O-dealkylase (EROD) activity in cultured cells or hepatic microsomes (Safe 1990). This induction segregates with other responses to TCDD and similar compounds because binding to the aryl hydrocarbon (Ah) receptor initiates signal transduction leading to the constellation of effects associated with Ah receptor agonists (Safe
EnvironmentalPCBs in Rats
1990; Ahlborg et al. 1994; McKinney and Waller 1994). Since the mechanism is known, it is also possible to estimate TEFs for Ah receptor-medicated toxic responses by calculating affinity for the Ah receptor (Kafafi et al. 1993). The less chlorinated and ortho-chlorinated congeners have low affinities for the Ah receptor and are poor inducers of CYP1A1; however, they induce CYP2B and have a profile of hormone and neurotransmitter dis~ption distinct from the '%0planar" Ah receptor agonists (Li et al. 1994; Ness et al. 1993; Seegal and Shain 1992; Soontornchat et al. 1994). In spite of their lower potencies, these congeners are present in far greater amounts than are coplanar CBs (Safe et al. 1987; Hansen 1987, 1994). It should be possible to predict potencies for some non-Ah receptor actions based on phenobarbital-type induction of pentoxyresorufin-O-dealkylase (PROD) activity (Safe 1994). Polychlorinated biphenyl-contaminated soils are of concern because of potential direct contact, mobilization by windblown dust, and contributions to atmospheric loads. Because of different physical/chemical properties, individual CBs will distribute unequally amongst the various environmental matrices (Hansen 1994). There is limited data on the composition and toxicity of soil-borne PCBs, so studies were initiated to determine a profile of responses and whether these responses could be predicted by TCDD equivalents. Air, subsurface soil, and superficial dust and debris from the small, but highly contaminated, Sangamo Landfill in southern Illinois (USEPA 1991) were sampled to determined potential toxicities. Short-term responses, more closely reflecting the initial actions of the toxicant(s), are most useful when attempting to differentiate potential subtle differences in toxicities. A short-term bioassay, using prepubertal female rats, has been able to detect multiple toxic effects, such as estrogenicity, P450 enzyme induction, and thyroid hormone depletion, of some ortho-chlorinated CBs and Aroclor® mixtures (Jansen et al. 1993; Li et al. 1994; Li and Hansen 1995; Soontornchat et al. 1994). The present study uses this short-term bioassay to describe enzyme induction, thyroid hormone depletion, and hematological effects of the Sangamo Landfill soil extract in an attempt to detect some of the actions and define the net potency of the toxicants within the extract.
335
hexane (A:H) and homogenization. The solvent was decanted and vacuum filtered and the extraction was repeated with 2 x 50 mL A:H. The soil was transferred to a teflon centrifuge tube and the extraction flask rinsed with 25 mL hexane into the tube; after centrifugation and solvent decanting, the procedure was repeated with an additional 25 mL hexane. The pooled extracts were dried over sodium sulfate and vacuum-concentrated at 57°C. During concentration of extracts, a dark oil was desolvated and the extracts were cleaned by slurry with 2 g 1% deactivated Florisil® (Erickson 1986). Anhydrous sodium sulfate was added until caking ceased (3-5 g) and the contents were filtered over sodium sulfate. The flask and Florisil-oil were extracted with 50 mL hexane, which was pooled with the previously decanted extracts. Solvent was exchanged for hexane and the extract was cleaned by alumina column chromatography (5 g 3% deactivated alumina, eluted with 50 mL hexane). The soil extract required reconcentration and a second alumina column before the concentrate was free of visible black oil. The refined extract was concentrated to 52.0 mL in hexane and aliquots were stored at 4°C in amber vials with teflon caps. Appropriate dilutions were semiquantitatively analyzed by EC-GLC to permit initiation of toxicity studies. Separate aliquots were transferred to other laboratories for PCB specific congener analysis, herbicide and insecticide analysis, and organometallic analysis. For specific congener analysis at the New York State Department of Health Wadsworth Laboratories (NYSDH), the aliquots were analyzed by EC-GLC (Bush et al. 1994). At the Illinois Hazardous Waste Research and Information Center, Hazardous Materials Laboratory (HWRIC), the samples were diluted and analyzed directly by GLC, but the effluent was split between the EC detector and an ion trap mass spectrometer (MS). The initial semiquantitative screen by EC-GLC estimated 64 mg/mL electron-capture detectable compounds. The NYSDH analysis determined a total PCB congener concentration of 38.6 mg/mL. The HWRIC EC + ion trap determinations at 2 sample dilutions averaged 52.5 mg/mL. The comparison of major congeners detected by both laboratories is presented in Table 1. Placing double weight (2 assays) on the HWRIC samples, a reliable estimate of 47.9 mg/mL PCB in the extract was determined. As expected, the initial serniquantitative screen for electron-capture detectable components was 33% higher than the mean for specific PCB congener analysis. The NYSDH analysis was 20% below the mean, while the HWRIC analyses were 9% higher. At similar PCB levels, spiked soil sample analysis by 20 reference laboratories were within 30% of each other (85 --- 26 ~g/g), while 129 accredited laboratories analyzed the same samples with 46% deviation (70 ± 32 Ixg/g) (Kimbrough et al. 1992).
TCDD Equivalents
Materials and Methods
Sample Collection, Extraction, and Analysis The Sangamo Landfill had been inactive since 1964 and access was limited when it was placed on the National Priorities List in 1984 (USEPA 1991). In 1992, dust and surface debris were removed from the proposed collection site by whisk broom; the next 5-10 mm of soil were removed by scraping with trowels, and finally, subsurface soil beneath the dust and debris was collected and sieved (No. 10) into a precleaned stainless steel bucket. Initially, 5 separate 10 g aliquots of soil were extracted with 3 x 20 mL acetone:hexane (1:1, v:v). These were semiquantitatively analyzed by electron capture (EC) gas-liquid chromatography (GLC) on a 60 m DB-5 capillary column, temperature programmed from 175°C to 275°C at 3°C/minute. The 5 soil samples were of nearly identical congener composition. Subsequently, 50 mL acetone was added to 100 g of soil (11% moisture) followed by mixing and addition of 50 mL 1:1 (v:v) acetone:-
The potential for TCDD-like actions of the mixture was determined using TCDD TEFs determined by Safe (1990), those calculated by Kafafi et al. (1993), and those generally agreed upon following a European conference (Ahlborg et al. 1994). The congener compositions of several Aroclors were determined by NYSDH, using the same methods as for the landfill extract, and TCDD equivalents were assigned to the Aroclors. The weight-based (rather than molar) TCDD equivalents of the soil extract were 0.78~).81 times that of Aroclor® 1248, and 2.4--2.7 times that of Aroclor 1242® (Table 2). Such calculated TCDD equivalents for Aroclor mixtures may not provide precise predictions of potencies due to various interactions (Harris et al. 1993; Hansen 1994; Safe 1994); nevertheless, a useful ranking is achieved and relative TCDD and nonplanar potencies can be further delineated by measuring EROD and PROD induction, respectively (Safe 1994). Animals, Dose Preparation, and Dosing Sprague-Dawley breeder rats were obtained from Harlan (Indianapolis, IN). Pups were culled to 8-10 animals per litter on the day of birth (day
336
L.G. Hansen et al.
0) and were weaned at 20 days of age. Doses were based on a nominal body weight of 0.05 kg on day 20 and calculated from the value of 48 mg/mL total PCB in the extract. Female pups were injected ip with soil extract dissolved in 0.1 mL corn oil or corn oil alone between 1:00 and 2:00 p.m. on day 20 and day 21. A negative control (corn oil) was included for each litter along with as many representative dose groups as the number of females would permit; therefore, there were 2 more control rats than in each of the 3, 12, 24, 36, 48, 72, and 96 mg/kg treatment groups (5 animals/group).
Data Analysis
Necropsy, Tissue Processing, and Measurements
Extract Residues
Rats were killed by decapitation between 9:00 and 11:00 a.m. on day 22. Blood was collected immediately after decapitation, 0.5 mL in an EDTA-treated syringe for clinical hematology and 0.5 to 1.0 mL from the cervical stump which was allowed to clot. Serum was separated by centrifugation and stored at -20°C until analyzed for T4 concentrations. Uteri were excised, trimmed of fat and connective tissue, and weighed. Livers were perfused in situ with ice-cold 0.05 M TrisHC1 + 0.15 M KC1 buffer (pH 7.4) as soon as the uteri were removed. Livers were excised, blotted on tissue paper, and weighed followed by homogenization in the same buffer. Kidneys and adrenal glands were removed and weighed. A smear was prepared from the unclotted blood and the remainder was diluted for autoanalysis in a Sismax F800 cell counter. The smear was autostained with Wright's stain for differential counts of a minimum of 1% of the total cell population. Thyroid glands were removed and immediately fixed in 10% neutral buffered formalin, processed routinely for histology, and stained with hematoxylin and eosin. Colloid area was quantified utilizing an automated computerized system (Videometric 150, American Innovision, Inc). A single cross section was evaluated for each animal, 20 follicles being selected by pre-determined grid location (Weibel 1979) and measured by calculating the area enclosed by the apical surface of follicular cells. The larger, more inactive follicles along the periphery of the gland were excluded from the analysis (Capen et al. 1991; Ness et al. 1993). Epithelial cell heights (3/follicle) of the same follicles were measured using the same system.
The soil extract used in the toxicity tests contained 48 mg/mL total CB congeners. Although the refined extract may not contain the exact proportions of each contaminant in the soil, it should be representative of the halogenated aromatic composition. 2,4,4'-triCB (CB 28) was by far the dominant congener in subsurface soil (Table 1). Unresolved CB 77/110 (3,3',4,4'tetraCB/2,3,3',4',6-pentaCB) was quantitated by GC-MS, which revealed 25% of coplanar CB 77 relative to CB 110 (Table 1), similar to that of Aroclors ® 1242 and 1248 (Duinker et al. 1988). Lower chlorinated ( < CB 101) congeners dominated and it is reasonable to conclude that some anaerobic dechlorination (Brown et al. 1987; Rhee et al. 1993) occurred in the very moist subsurface soils. Chlorobiphenyl 28 has previously been shown to be very recalcitrant to this type of dechlorination (Rhee et al. 1993). The only currently accepted means of describing PCB residues, other than total PCB content, is to calculate total equivalency to TCDD (TCDD equivalents) (Safe 1990, 1994; Ahlborg et al. 1994). The refined soil extract had a total TCDD equivalent between that of Aroclor 1242 and that of Aroclor 1248 (Table 2). Polychlorinated naphthalenes (PCNs), dibenzofurans (PCDFs), and dibenzodioxins (PCDDs) have also been demonstrated to be present at lower concentrations (USEPA 1991). They have not yet been quantitated, but their presence would add to the TCDD equivalent of the soil extract, probably to the extent of exceeding that of Aroclor® 1248. The presence of PCNs could partially account for the higher estimates in the initial semiquantitative screens. A modest amount (125 Ixg/mL) of 2,2-bis-p-chlorophenyl-1,1-dichloroethylene (p,p'-DDE) was found in the extract (Table 1). No other insecticides or herbicides were found at detectable concentrations. The possibility of bioalkylation of heavy metals was considered, so all extracts were screened for mercury, lead, and tin on a GC equipped with an atomic emission detector; no response was observed.
Microsomal Preparation and Biochemical Assays The crude liver homogenate was centrifuged at 10,000 g for 15 min at 4°C. The supernatant was spun at 105,000 g for 60 min at 4°C. The pellet from the last centrifugation was resuspended in a buffer (0.05 M Tris-HC1, 20% glycerol (v/v), 1 mM EDTA; pH 7.4) and stored at -80°C until assayed (Li et al. 1994). 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation were determined by a modification of the method of Pohl and Fouts (1980). In brief, the reaction mixture contained 5 mM MgC12, 1 mg bovine serum albumin, microsomal suspension (200 to 500 ~g of microsomal protein for EROD and 400 to 800 p~gfor PROD), 2.5 p,M ER or 10 p,M PR in 0.05 M Tris-HCl (pH 7.4), and a NADPHgenerating system (0.8 mg NADP+, 1.5 mg glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase). The reaction was initiated by adding the NADPH-generating system and stopped by the addition of 2 mL of methanol. The reaction was carried out 4 min for EROD and 10 rain for PROD at 37°C. The formation of resorufin was determined by measuring sample fluorescence relative to a known amount of resorufin with excitation at 550 nm and emission at 585 nm in a Perkin-Elmer 203 Fluorescence Spectrophotometer. Microsomai protein was determined by a modification of the Lowry method (Guengerich 1982) using bovine serum albumin as a standard. All samples were run in duplicate. Serum total T4 was determined using a radioimmunoassay (RIA) kit (Coat-A-Count®) purchased from Diagnostic Products Corporation (Los Angeles, CA). The limit of the detection for total T4 was 0.25 p,g/dL. All samples were run in duplicate.
All data were analyzed by one-way analysis of variance (ANOVA). If a significant result was found, post hoc comparisons between treatments and control were then conducted by Dunnett's t-test.
Results
Rat Responses Relative liver weights increased significantly at doses between 12 and 72 mg/kg, but seemed to plateau between 72 and 96 mg/kg (Table 3). Although not statistically significant, microsomal protein increased in parallel and plateaued or declined at the highest dose. The specific activities of cytochrome P450 1A1 (as EROD) and P450 2B (as PROD) increased abruptly at doses above 3 mg/kg (Table 3). Ethoxyresorufin O-dealkylase activity increased and plateaued at lower doses than did PROD. Selected microsomes were reassayed at lower protein contents, but the specific activity remained the same; thus, the plateau was not due to exceeding the capacity of the enzyme assay
Environmental PCBs in Rats
337
Table 1. Major polychlorinated biphenyls (PCB) congeners in landfill soil extracts analyzed by the New York State Department of Health (NYSDH) and the Illinois Hazardous Waste Research and Information Center (HWRIC). IUPAC numbers as per Ballschmiter et al. (1987) IUPAC No. 6 19 18 15 + 17 24 + 27 16 + 32 26 25 28 + 31 33 22 45 52 49 47 + 48 44 37 + 42 + 59 41 + 64 + 71 40 74 + 94 70 66 + 95 56 + 60 92 84 101 99 97 81 + 87 DDE 136 77 + 110 82 + 151 135 118 149 146 132 + 153 105 141 138 158 126 182 183
Chlorine pattern
NYSDH
Ring A
Ring B
2 2,6 2,5 4 2,4 2,3,6 2,6 2,3 2,4 2,5 2,4 2,4 2,5 2 2,3 2,3,6 2,5 2,4 2,4 2,4,5 2,3 3,4 2,3 2,3,6 2,3,4 2,3,6 2,6 2,3 2,4,5 2,3,5 2,5 2,4 2,3,6 2,3 2,3,4 2,3,5 2,3,6 2,4,5 2,4,5 2,4,5 3,4,5 2,3,4
3 2 2 4 2
2,3,6 3,4 2,3,6 2,3,4 2,3,5,6 2,3,5 2,4,5 2,3,6 2,3,5 2,3,4 2,4,5 2~3,4 2,3,4,5 2,3,4 2,3,4,6 3,4,5 2,3,4,5 2,3,4,6
~ 3 2 6 3 3 4 4 3,4 4 2 2,5 2,5 2,4 2 2,5 4 2,4 3 2 4 3,4 2,3 4 2,6 3,4 3,4 2,5 3,4 4 3,5 2,3 2,5 2,4 2,3 4 2,5 2,3,6 3,4 3,4 2,3 2,5 2,3,6 3,4 2,4,5 2,4,5 2,3,6 2,4,5 3,4 2,5 2,4,5 3,4 3,4 2,4,6 2,4,5
Ixg/mL 349 412 1401 857 NR b 539 NR 2118 NR 404 109 6552 1278 1102 1483 467 3004 1692 591 NR 2031 1837 NR NR NA 1477 NA 539 529 0 1231 2337 NR 416 NR NA NA 941 245 247 NR 193 125 60 1413 NR 104 NR 51 383 322 45 344 NR 100 84 454 49 NA NA 45
HWRIC % Total
ixg/mL
0.90 1.07 3.63 2.22
NA a NA 2900 390 1000 NA NR 3400 NR NA NA >5100 NR 500 3000 800 2700 1958 >665 NR 2430 1500 750 NA 3490 NR NR >500 >500 100 81
1.39 5.48 1.04 0.28 16.96 3.31 2.85 3.84 1.21 7.78 4.38 1.53 5.26 4.76
3.82 1.40 1.37 0.00 3.19 6.05 1.08
2.44 0.63 0.64 0.50 0.32 0.16 3.66 0.27 0.13 0.99 0.83 0.12 0.89 0.26 0.22 1.18 0.13
0.12
% Total
5.70 0.77 1.97
6.69
10.03 0.98 5.90 1.57 5.31 3.85 1.31 4.78 2.95 1.48 6.86
0.98 0.98 0.02 4.63 5.05 1.75 5.07 0.39 0.83 2.04 1.01 0.88 0.01 1.07 0.20 0.67 2.54 0.38 0.30 1.65 1.38 0.22 0.22 1.29 0.98 0.32 1.55 0.21 0.03 0.20 0.16
338
L.G. Hansen et al.
Table 1. Continued
IUPAC No. 128 + 167 174 177 156 + 171 180 169 170 + 190 209 SUM
Chlorine pattern
NYSDH
Ring A
Ring B
2,3,4 2,4,5 2,3,4,5 2,3,5,6 2,3,4,5 2,3,4,6 2,3,4,5 3,4,5 2,3,4,5 2,3,4,5,6 2,3,4,5,6
2,3,4 3,4,5 2,3,6 2,3,4 3,4 2,3,4 2,4,5 3,4,5 2,3,4 3,4 2,3,4,5,6
txg/mL
HWRIC % Total
~g/mL
% Total
87
0.22
0.30
80 42 34
0.21 0.11 0.09
152 NR 151 62 94 39 280
NR
NR 142 NA 75 NR NA 38419
0.37
1
0.19
140 NR 1
99.4
50862.8
0.30 0.12 0.19 0.08 0.55 0.00 0.28 0.00 98.82
aNot analyzed bNot resolved from congener listed previously; value represents total of both CNo C1 substitution on ring B
system. It was thought that the combination of specific activities, amounts of microsomal protein, and liver weights might exaggerate differences; nevertheless, total activity per liver reached a firm plateau with no evidence for further increases or declines at higher doses for either enzyme. The relative weights of uterus and kidneys did not vary among the dose groups; relative adrenal gland weights seemed to be trending higher, but the differences were irregular and not significant. Serum total T4 (TT4) declined in a dose-dependent manner to less than 50% of control values (Table 4), but no further decline was observed between 72 and 96 mg/kg. Some of the thyroid glands were fixed and processed to evaluate thyroid follicular morphology. Even though TT4 declined more or less linearly, mobilization of storage forms was demonstrated at doses lower than those causing significant decreases: the average area of thyroid follicular colloid declined at the lowest dose and then remained constant (Table 4). The average height of follicular epithelial cells likewise increased at the lower doses to what was apparently the maximum response possible. Hematological parameters are presented in Table 5. Minor short-term changes in the rapidly developing hematopoietic system would be difficult to demonstrate and it was initially believed that no consistent trends were emerging. Nevertheless, by combining the limited data into dose ranges, some trends are suggested, indicating that it would be inappropriate to deny responses. The number of nucleated (young) erythrocytes decreased with dose and the mean corpuscular volume (MCV) appeared also to be decreasing; however, at the higher doses, the cell volume increased abruptly. Although there were only 4 animals included in the high dose group (1 at 48, 2 at 72, and 1 at 96 mg/kg), the accompanying decline in mean corpuscular hemoglobin concentration (MCHC) suggests that some type of osmotically driven cell swelling may have occurred. There is also a trend toward neutrophilia in which the highest dose group contained a significantly greater percent and number of neutrophils than the lowest dose group. These possible effects require further clarification.
Discussion
Even though doses were purposely limited to moderate levels, maximum EROD activity appeared to plateau near 1900 pmole/ min/mg microsomal protein at low doses (Table 3). Under the same conditions, 400 mg/kg Aroclor® 1242 induced EROD activity to a very similar 1650 pmole/min/mg (Li et al. 1994). In adolescent male Wistar rats, Aroclor® 1242 induced EROD activity to the same level at 160 mg/kg, but a maximum activity of 5000-7000 pmol/min/mg was achieved with 2000 mg/kg Aroclors ® 1232, 1242, 1248, and 1254 15 days following a single ip injection (Hams et al. 1993). The lower maximum under the conditions used in the current study represents the limited time (44 hours) and possibly, limited responsiveness of the weanling rat liver. However, higher EROD induction (2800 pmol/min/mg) was achieved in the same strain of weanling rats exposed to a thousand-fold lower dose of TCDD in utero (Seo et al. 1995), so it is probable that the time allowed did not permit full expression of the induced enzyme. Nevertheless, the high EROD induction at all but the lowest dose indicates substantial Ah receptor-mediated responses to the landfill soil extract. Maximum PROD activity appears to be underestimated to an even greater extent under the conditions of the bioassay. In a previous study using these younger female rats, PROD activity was 17.3 pmol/min/mg (5 times control levels) 44 h after the first of 2 doses of 200 mg/kg Aroclor® 1242 (Li et al. 1994); in the current study, it plateaued at 12-13 pmol/min/mg after more moderate doses of soil extract (Table 3). In older male rats permitted 15 days to express the enzymes, PROD activity of 200-500 pmol/min/mg was achieved with 160-2,000 mg/kg of several Aroclors ®, including Aroclor® 1242 (Hams et al. 1993). CYP2B induction is somewhat slower and less responsive than that of CYP1A1, so the apparent plateau of PROD activity well below the apparent biological limit of inducibility probably represents the maximum responsiveness of these prepubertal livers within the short time period. Nevertheless, significant induction at moderate doses coupled with the lack of
Environmental PCBs in Rats
339
Table 2. TCDD ~ equivalents calculated for Aroclor 1242, 1248, and Sangamo Landfill soil extract using three different TEF systems from Safe (1990), Kafafi et al. (1993) and Ahlborg et al. (1994) Percent Total PCB IUPAC No. 1 3 4 6 7 8 16 17 18 19 22 24 25 26 28 31 33 40 42 44 45 47 49 52 59 60 64 70 77 87 94 97 99 101 105 118 128 129 130 134 136 138 141 146 149 151 153 158 170 171 172 174 176 177 179 180 183 185 187 194
TEF a
TEF b
TEF ~
10-6 + 10 + 9 + 5 +32
10 - 6
+27 10 - 6 10 - 6 10 - 6
10 - 6 10 - 6
+48
+42+39 +56
10 -5 10 - 6
+110 d
10 -2
+74
10 - 3
5 × 10 - 4
10 -5 10 - 6
+167
+132 +190
10 -3
10 -4
10 - 3
10 - 4
2 × 10 -5
10-5
2 × 10 -5
10 -6
2 × 10 -5 2 × 10 -5 2 × 10-5
10 -5 10 -5 10 -5
10 -4 10 -~ 10 -5
10 -4
10 -5 + 163
2 x 10 -5
10 -5
2 × 10 -5
10 -4
10 - s
Aroclor® 1242
Aroclora 1248
Soil extract
0.00 0.00 3.30 2.20 0.90 7.80 5.70 5.80 8.20 0.70 2.50 0.80 0.20 0.90 10.30 5.70 7.60 0.80 4.40 4.10 1.10 2.20 3.20 5.90 3.40 2.00 2.50 2.30 1.40 0.30 0.90 0.40 0.40 1.30 0.20 0.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.60 0.30 0.00 1.50 2.50 2.20 4.60 0.20 1.30 0.20 0.00 0.40 10.20 0.90 2.60 1.20 8.40 7.10 1.20 3.50 5.50 11.60 4.40 4.90 3.80 4.50 4.60 0.90 1.40 1.30 1.30 3.80 0.50 1.30 0.00 0.00 0.00 0.00 0.00 0.30 0.00 0.00 0.30 0.60 0.10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.90 0.00 0.00 5.48 2.22 3.63 1.07 2.85 1.39 0.28 1.04 16.96 3.31 0.00 1.40 6.05 5.26 2.21 1.53 4.38 7.78 4.76 1.08 3.82 3.19 3.66 0.50 1.37 0.64 0.63 2.44 0.26 0.99 0.22 0.00 0.00 0.00 0.16 1.18 0.22 0.12 0.83 0.27 0.89 0.13 0.19 0.09 0.00 0.21 0.00 0.1 l 0.00 0.37 0.12 0.00 0.00 0.00
340
Table
L.G. Hansen et al. 2. Continued Percent Total PCB Aroclor®
IUPAC No.
TEFb
TEFa
TEF~
10 -6 10-6
195 196 199 200 201 206 TCDD equivalental TCDD equivalentb~ TCDD equivalentc~
10 -6
1242
Aroclor® 1248
Soil extract
0.00 0.00 0.00 0.00 0.00 0.00 0.0033
0.00 0.00 0.00 0.00 0.00 0.00 0.01101
0.00 0.00 0.00 0.00 0.00 0.00 0.00863
0.00039
0.00118
0.00093
0.00019
0.00064
0.00052
aSafe 1990 bKafafi etal. 1993 CAhlborg et al. 1994 ORatio of CB 77 to CB 110 for Aroclors® 1242 and 1248 were 1:4 based on Duinker et al. 1988 eTCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin Table 3. Liver weights, microsomal protein contents, and microsomal enzyme activities in immature female rats treated with landfill soil extracts Dose (mg/kg) 0 3 12 36 48 72 96
n
Relative liver weight (%)
Microsomal protein content (mg/g liver)
ERODb (pmole/min/mg protein)
PRODc (pmole/min/mg protein)
7 5 5 5 5 5 5
4.5±0.1 a 4.5 ± 0.2 5.1 - 0.2 5.3 ± 0.1"** 5.3 ± 0.1"* 5.9 ± 0.2*** 5.4 ± 0.2***
11.0±0.7 10.4 ± 0.3 10.5 ± 0.9 12.3 ± 1.3 13.7 - 1.1 13.9 - 1.6 11.7 ± 2.0
85.6± 394.5 ± 1577.0 ± 1922.6 ± 1871.4 ± 1682.0 ± 1747.5 ±
3.3-0.6 4.5 ± 0.7 10.3 ± 1.3"** 10.9 ±- 1.4"** 12.8 ± 1.5"** 12.6 ± 1.5"** 12.9 ± 2.7***
21.9 79.4 205.4*** 178.7"** 188.4"** 150.6"** 277.7***
aMean ± SEM bEROD = ethoxyresorufin-o-dealkylase c P R O D = pentoxyresorufin-o-dealkylase ** :P < 0.05 forpost hoc comparison to control *** :P < 0.01 forpost hoc comparison to control
significant responses at the lowest dose suffices to confirm a greater-than-Aroclor 1242 TCDD equivalent (EROD) and suggest a lesser-than-Aroclor® 1242 nonplanar toxic equivalent (PROD). Further studies should be conducted to determine induction after longer times to determine if interactions within the complex mixture further limit the enhancement of EROD and PROD activities. At the higher doses, enhanced biotransformation may produce adequate levels of hydroxylated (Schmoldt et al. 1977) and/or methyl sulfonyl (Nagayama and Masuda 1993) metabolites to inhibit the measured activities of the induced P450s. The presence of readily metabolized substrates such as 2,2',5-triCB (CB 18) may also compete for the enzymes (Hansen 1979, 1987). Competing down-regulation of P450s by less potent 2,3,6-substituted congeners as seen with chronic exposure in laying hens (Hansen 1979) is less likely in this short-term exposure. Extended studies to confirm or deny attenuated activity compared to gene expression are necessary because these types of interactions may limit the potential utility of TEFs based on induced enzyme activities in the evaluation of complex environmental mixtures (Hansen 1987, 1994; Safe 1994).
Estrogenicity, as measured by the uterotropic response, is an action of Aroclor® 1242 and is also characteristic of lower chlorinated CBs such as CB 18 (Li and Hansen 1995) and of some nonplanar PROD inducers such as 2,2',4,4'-tetraCB, 2,2',5,5'tetraCB, and 2,2',4,4',5,5'-hexaCB (CBs 47, 52, and 153, respectively) (Jansen et al. 1993; Li et al. 1994; Soontornchat et al. 1994). The lack of a demonstrable estrogenic effect may also be due, at least in part, to interactions. Greater than 13% of the PCB content of the soil content was due to these congeners recently demonstrated to cause a uterotropic response in prepubertal rats. If closely related probable estrogens are included, this proportion more than doubles. The coplanar CB 77 does not cause this response and, in fact, antagonizes the uterotropic response of prepubertal female rats to exogenous estradiol as well as to Aroclor® 1242 (Jansen et al. 1993). Antiestrogenicity is associated with Ah receptor agonists (Krishnan and Safe 1993). The TCDD equivalent of the no-net-response 320 txg Aroclor® 1242 + 160 ixg CB 77 combination (Jansen et al. 1993) would be double that of Aroclor® 1242 alone and therefore slightly less than the TCDD equivalent of the soil extract; thus, it is not surprising that no uterotropic response was de-
Environmental PCBs in Rats
341
Table 4. Serum total thyroxine (T4), thyroid follicle colloid area, and follicular cell height in 22 day female rats receiving landfill soil extracts on days 20 and 21 Dose (mg/kg)
T4" (txg/dL)
0 3 12 36 48 72 96
2.4 2.1 1.9 1.5 1.3 0.9 1.0
-+ +-± ± ± ± ±
0.2 ¢ 0.1 0.3 0.2*** 0.2*** 0.1"** 0.2***
Colloid areab (tJ,m 2)
Cell heightb (Ixm)
1181 ND d 659 665 534 660 ND
9.4 ND 10.7 11.6 11.0 11.0 ND
± 143 ± ± -
41"** 46*** 88*** 67***
± 0.5 ± ± ± ±
0.6 0.3*** 0.2* 0.2**
an = 5 in all dose groups except n = 7 for control bn = 4 for morphometry results except n = 3 at 48 mg/kg group CMean ± SEM dNot determined * :P = 0.062 marginally significant forpost hoc comparison to control ** :P < 0.05 forpost hoc comparison to control *** :P < 0.01 forpost hoc comparison to control
Table 5. Clinical hematology parameters in 22 day female rats receiving landfill soil extracts on days 20 and 21 Dose range (mg/kg)
Control
3-12
24-36
48-72-96
n
4
5
4
4
Red Blood Cells Total (106) Nucleated (%) HB (gm%) PCV (%) MCV (fL) MCH (pg) MCHC (g/dL) White Blood Cells Total (103) Neutrophils (%) Neutrophils (#) Lymphocytes (%) Lymphocytes (#) Monocytes (%)
4.7 5.0 11.7 35.4 76.2 25.2 33.0 5.8 23.5 1347 72.8 4245 3.0
± + + + ± ± ±
0.4 a 3.l 0.2 0.9 3.8 1.7 0.6
± 0.8 ± 3.6 ± 219 ± 3.9 - 593 1.0
4.4 3.4 10.6 33.0 76.7 24.6 32.1 4.4 20.4 944.4 77.6 3416 1.8
+ ± ± + + ± -
0.4 1.4 0.5 1.8 5.3 1.6 0.3
4.9 1.8 11.l 35.0 70.8 22.5 31.8
± 0.6 ± 3.7 ± 263 ± 3.4 -+ 464 0.5
5.3 30.0 1603 66.5 3536 3.5
+ ± ± ± + ± ±
0.1 0.6 0.1 0.8 1.5 0.3 0.6
4.5 0.3 10.7 34.8 78.3 24.0 30.6
± 0.1 ± 2.8 ± 178 ± 2.8 ± 140 ± 0.3
7.3 36.0 2593 61.8 4502 1.5
+ -+ ± ± ±
0.2 0.3 0.2 0.3 4.1 1.1 0.6**
± 1.0 ± 3.1" ± 364** ± 3.3 ± 717 ± 0.9
aMean - SEM * LP = 0.062 marginally significant forpost hoc comparison to control ** :P < 0.05 forpost hoc comparison to control
tected in these rats in spite of the presence of a significant estrogenic component. Such a balanced neutralization of xenoestrogens has been cited as a reason for a low level of concern regarding these agents (S. Safe in Stone 1994), but failure to maintain the balance may unmask previously undetected effects (Hansen and Jansen 1994). It should be of interest to compare T C D D equivalents and estrogenicity of the other matrices from the landfill to determine if this balance may already be violated by some environmental residues. It is also possible that the uterine response may be attenuated in the rats because of the concurrent depletion of thyroid hormones. The diminished uterine responsiveness in hypothyroid rats (Kirkland et al. 1981; Eriksson and Freyschuss 1988) is one of the reasons for limiting the duration of exposure when attempting to detect multiple actions. Within the short duration of the bioassays, early decreases in circulating thyroid hormones are counteracted by mobilization of storage forms so that systemic hypothyroidism is delayed.
Although most PCBs appear to depress serum TT4 (Bastomsky et al. 1976; B y m e et al. 1987; Ness et al. 1993), the dominant effect is probably not due to Ah receptor agonists at the level of E R O D induction achieved in this study (Seo et al. 1995). In the Seo study using T C D D and coplanar PCBs, E R O D activity was 59% higher, but T4 depletion was much more modest. Exposure to 2 , 3 ' , 4 , 4 ' , 5 - p e n t a C B (CB 118) in utero can result in weanling rat TT4 as low as 10% of controls and a dose-dependent decrease in thyroid follicular colloid area to 700-800 izm2 (Ness et al. 1993). In these rats exposed to a complex PCB mixture, colloid area was reduced to 600 txm 2 with a more modest decline in serum TT4; moreover, higher doses and greater depletion of TT4 did not further reduce the colloid area (Table 4). Apparently, different combinations of mechanisms must be considered for different PCB mixtures. For the soil extract, colloid area decreased at doses which did not cause significant T F 4 depletion. The serum TT4 was probably restored by thyroglobulin mobilized from the follicular colloid (supported by increased height of the epithelial cells);
342
thus, the sensitivity of serum TT4 measurements for mixtures containing multi-ortho chlorinated congeners is reduced via this mobilization mechanism which seems to be attenuated following in utero exposure to mono-ortho congeners such as CB 118. Nevertheless, TT4 depletion by PROD-inducing congeners seems to be more effective than depletion initiated by Ah receptor agonists. In summary, we were able to detect toxic effects of a landfill soil extract on multiple biochemical and endocrine endpoints by using a short-term rat bioassay. The major effects of landfill soil extract on prepubertal female rats include hepatic microsoreal P450 enzyme induction, liver weight increase, depression of serum TT4, and changes in the morphology of thyroid follicles. On the other hand, even though a large proportion of proven and probable estrogenic CBs were present, the lower amounts of more potent antiestrogenic Ah receptor agonists and/or decreased responsiveness because of low serum TT4 levels may have antagonized the uterotropic response. Acknowledgments. This research was supported by the Illinois Department of Energy and Natural Resources, Hazardous Waste Research and Information Center Grant (HWRIC) 93-106. ECD specific congener analysis, ion trap GC-MS discrimination of co-eluting congeners, and GC-AES anlysis for heavy metals were conducted bY David Green and Jack Cochran, HWRIC. The authors also thank Ruthann Nichols (Clinical Pharmacology) for the TT4 analyses, Dave Pryor (Clinical Pathology) for the CBCs, and Dr. Thomas Eurell for assistance in interpreting the CBCs.
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