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Short-term effects of the tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on I-compounds in liver, kidney and lung DNA of male Sprague-Dawley rats
Toxicology, 68 (1991) 275-289 Elsevier Scientific Publishers Ireland Ltd.
275
Short-term effects of the tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on I-compounds in liver, kidney and lung DNA of male Sprague-Dawley rats Raghu G. Nath, Erika Randerath and Kurt Randerath Division of Toxicology. Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) (Received March 27tb, 1991; accepted June 12th, 1991)
Summary The effects of a tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on l-compounds (tissue, species and sex dependent D N A modifications that increase with age in untreated rodents) were studied by 32p-postlabeliffg in male Sprague-Dawley rat liver, kidney, and lung DNA. Aroclor 1254 was dissolved in corn oil and intraperitoneally (i.p.) injected (2 x 500 mg/kg, 2 weeks apart) into 3-month-old rats. Control rats were given corn oil. Groups of 3 animals were sacrificed at 2 and 6 weeks after the second injection of corn oil or Aroclor 1254. At both time points Aroclor 1254-treated rats had significantly lower body weights and higher liver weights while kidney and lung weights were unaffected. Thymidine incorporation into liver and lung D N A was significantly increased at both time points, while kidney D N A showed a small decrease at 2 weeks. Treatment resulted in significant reductions (ranging from 29 to 100'%) of each of nine liver I-spots at 2 and 6 weeks. In treated rats there was no decrease in kidney I-spots at 2 weeks, while the levels of only two out of ten kidney spots were reduced by 42-91'7o at 6 weeks. At 2 weeks three out of seven and at 6 weeks four out of seven lung I-spots were lowered by 51-100% in the Aroclor 1254-treated rats. Thus the effects decreased in the order liver > lung > kidney. Since Aroclor 1254 has been reported to be a tumor promoter in liver and lung but not kidney, these results suggest a correlation between organ specific promotion of carcinogenesis by Aroclor 1254 and the reduction of D N A l-compounds.
Key words: Aroclor 1254; Carcinogenesis; D N A synthesis, I-compounds: Polychlorinated biphenyls; T u m o r promotion
Correspondence to: Kurt Randerath, Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, U.S.A. Abbreviations: Ah, aryl hydrocarbon; C, central; D, direction; [3H]TdR, [methyl-3H]thymidine; Icompounds, D N A modifications which tend to increase with age in tissues of untreated animals: L, lower; PCB, polychlorinated biphenyl; PEI, polyethyleneimine; RAL, relative adduct labeling, a measure for the level of covalent modifications of DNA; T C D D , 2,3,7,8-tetrachlorodibenzo-p-dioxin: TLC, thin-layer chromatography; U, upper. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
276 Introduction
Novel covalent DNA modifications termed I(indigenous)-compounds have been detected by the 32p-postlabeling assay for DNA adducts in various tissues of untreated rodents [1,2]. I-compounds tend to gradually accumulate with increasing animal age, and their profiles and levels depend on species, sex, tissue, and diet [2,3]. I-compounds are presumably formed by the interaction of DNA with endogenous electrophilic metabolites of hormonal [2] or dietary [4] origin. Procedures known to induce liver cancer, i.e. feeding a choline devoid diet [5] or treatment with nonmutagenic hepatocarcinogens such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [6,7], carbon tetrachloride [8], and peroxisome proliferators [9] result in significantly diminished I-compound levels in rodent liver. Rat liver I-compound levels reach a plateau at about 16-24 months of age, remaining unchanged for the most part as the animals grow older [10] and become more susceptible to spontaneous tumorigenesis [11]. Strains of mice highly susceptible to spontaneous hepatomas have relatively lower levels of liver l-compounds than less susceptible strains [12]. I-compounds were found virtually absent from a spectrum of Morris hepatomas [13]. Thus a multitude of observations suggests that reduced I-compound levels are associated with, and may perhaps contribute to carcinogenesis. Polychlorinated biphenyls (PCBs) are chemically stable environmental contaminants now widely distributed in every component of the global ecosystem. The toxic effects of PCBs include thymic atrophy, a wasting syndrome, immunotoxic responses, hepatotoxicity, and chloracne [14]. Available evidence does not conclusively show a greater cancer risk in humans exposed to PCBs [15]. Since highly chlorinated PCBs are poorly metabolized in the body they are not readily activated to reactive metabolites capable of covalent binding to DNA and tissue macromolecules. As a result they are likely to act as tumor promoters rather than initiators. Aroclor 1254 is inactive in mutagenicity tests using the Ames Salmonella assay [16]. Chronic feeding of high doses of Aroclor 1254 induces liver tumors in mice [17] and rats [18]. The tumor promoting effects of PCBs are studied mostly in Sprague-Dawley and F344 rats as well as various strains of mice. Aroclor 1254, Kaneclor 500, and other PCB mixtures (unspecified components) are commonly employed in promotion studies [14]. Studies using the two-stage hepatocarcinogenesis model clearly indicate the tumor promoting effect of Aroclor 1254. In partially hepatectomized male SpragueDawley rats given diethylnitrosamine, one dose of Aroclor 1254 increases the incidence of gamma-glutamyltranspeptidase-positive foci [19], while in rats not treated with diethylnitrosamine, Aroclor 1254 treatment does not increase the incidence of such foci indicating the lack of an initiating effect. In mice initiated with dimethylnitrosamine, Aroclor 1254 increases the size of hepatic tumors and foci and also the incidence of lung tumors [20]. In another study [21], when the tobaccospecific carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone was administered to pregnant mice and the progeny were given a single dose of Aroclor 1254 the incidence of hepatic tumors significantly increased. The tumor promoting effect of Aroclor 1254 in male Sprague-Dawley rat liver was shown not to be due to polychlorinated dibenzofuran impurities [22].
277 Some of the biologic and toxic effects of PCBs resemble those exhibited by polychlorinated dibenzo-p-dioxins. Previous reports showed T C D D to be a hepatocarcinogen in female rats [23] and also a tumor promoter [24]. Reports from our laboratory have shown that T C D D selectively decreases hepatic I-compound levels in the female rat [6,7]. T C D D is not carcinogenic to the kidney [23] and the kidney I-compound level is not affected in the female rat [6,7]. Aroclor 1254, like TCDD, is known to competitively bind to the specific aryl hydrocarbon (Ah) receptor [25]. In the present study we investigated the effect of Aroclor 1254 on liver, kidney, and lung I-compounds in male Sprague-Dawley rats. Materials and methods
Materials [methyl-3H]thymidine ([3H]TdR, 71 Ci/mmol in 70% ethanol) and aqueous [32p]phosphate (HCI-free; >300 mCi/ml) were purchased from ICN Radiochemicals (Irvine, CA). The sources of all other biochemical and chromatographic materials used for adduct analysis have been previously reported [26-28]. Animals and treatment Male Sprague-Dawley rats (3 months old, 230-260 g) were purchased from Harlan Sprague-Dawley, Inc. (Houston, TX). The rats were housed three per cage and fed Wayne Lab Chow ( M R H 22/5 Rodent Blox) and tap water ad libitum. Aroclor 1254 (Analabs, New Haven, CT) was injected intraperitoneally (i.p.) at a dose of 500 mg/kg. To administer this dose the compound was dissolved in corn oil (Sigma, St. Louis, MO) to make a 400 mg/ml solution and i.p. injected in a volume of 0.125 ml/100 g body weight. Twelve rats were divided into four groups of three animals each. Rats of groups 1 and 3 (control) received two injections of corn oil (0.125 ml/100 g) 2 weeks apart and the rats of groups 2 and 4 (experimental) were given two injections of Aroclor 1254 (500 mg/kg) 2 weeks apart. Two weeks after the second injection, rats of groups 1 and 2 were given three i.p. injections each of [3H]TdR (150/~Ci/kg) every 2 h starting at 1000 h. The rats were sacrificed 2 h after the third dose of [3H]TdR, the liver, kidneys, and lungs were removed, cleaned, weighed, minced, and frozen at -80°C until DNA isolation. Six weeks after the second dose of corn oil or Aroclor 1254, rats of groups 3 and 4 were sacrificed and tissues removed after similar [3H]TdR treatments. DNA isolation and 32p-postlabeling DNA was isolated from liver, kidneys, and lungs of three individual animals by solvent extraction combined with enzymatic digestion of proteins and RNA [29]. The amount of radioactivity (3H) incorporated into DNA was determined by counting 50 #g DNA from each individual organ in a Packard Scintillation Counter (Tricarb 4000 Series) using 10 ml Aqueous Counting Scintiilant (ACS, Amersham Corporation, Arlington Heights,lL). DNA from each sample was analyzed in duplicate by a nuclease PI enhanced 32p-postlabeling assay [28]. Briefly, 10 /~g DNA was hydrolyzed to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase, followed by
278 incubation with nuclease P1 to 3'-dephosphorylate normal nucleotides [28]. Digestion was terminated by the addition of 3.0 /~1 of 2-[N-cyclohexylamino]-ethane sulfonic acid (CHES) buffer (0.5 M, pH 9.6), then the mixture was evaporated to dryness and reconstituted in 10 t~l distilled water. The adducts were labeled with excess carrier-free [3,-32p]ATP (3500-5000 Ci/mmol), spotted on a polyethylene±mine (PEI)-cellulose thin layer and developed overnight in 2.3 M sodium phosphate, pH 5.75 (D1) to remove excess ATP and normal nucleotides [27,28]. The non-polar Icompounds retained in the origin area (L, lower cut) and more polar I-compounds which migrated out of the origin [center (C) and upper (U) cuts] were contacttransferred to three separate fresh PEI-cellulose sheets and resolved by twodimensional thin-layer chromatography (TLC) as previously described [30]. Age dependent increases were not detected for the majority of I-compounds, probably because the 4 week observation period was too short. 32p-labeled fractions were identified as I-compounds by TLC comparison with previously characterized I-compounds in male rats [3,5,7,30]. 32p-labeled l-spots were located by screenenhanced autoradiography, excised, and subjected to scintillation (Cerenkov) assay for estimation of relative adduct labeling (RAL) values [30]. These values were calculated from the net count rates of spots, the specific activity of ATP, and the amount of DNA as described [28,30] and were expressed as RAL x 109 values, representing a minimum estimate of the number of modified nucleotides in 109 DNA nucleotides. Means ± standard deviations (S.D.) of RAL values were evaluated for significant differences by Student's t-test. Results
Tissue and body weights Table I compares the mean ( + S.D.) body weights, organ weights, and organ/body weight ratios from corn oil and Aroclor 1254-treated rats. Treated rats had significantly lower body weights than control rats at 2 (76% of control) and 6
TABLE 1 EFFECT
OF AROCLOR
SPRAGUE-DAWLEY
1254 O N B O D Y , L I V E R , K I D N E Y ,
AND LUNG WEIGHTS
IN M A L E
RATS* 6 Weeks
Time point
2 Weeks
Treatment
C o r n oil
A r o c l o r 1254
C o r n oil
A r o c l o r 1254
356.30 4- 12.60 12.91 ± 1.42
271.30 ± 8.50 a 17.85 ± 1.34 b
408.30 ± 28.00 11.85 ± 1.05
294.30 .4- 25.70 a 19.87 4. 1.35 b
B o d y wt (g) Liver wt (g) L i v e r / B o d y wt (%) K i d n e y wt (g) K i d n e y / B o d y wt (%) L u n g wt (g)
3.62 2.61 0.73 1.53
± 4± 4-
0.44 0.15 0.03 0.09
L u n g / B o d y w t (%)
0.43 ± 0.02
6.58 2.27 0.84 1.70
44:t: 4-
0.46 b 0.29 0.08 0.43
0.63 4- 0,16
2.90 2.48 0.61 1.90 0.47
+ + ± .44-
0.06 0.21 0.01 0.65 0.13
*All g r o u p s c o n s i s t e d o f 3 a n i m a l s . a,bSignificantly different f r o m c o r n oil c o n t r o l s ( P < 0.001 a n d 0.01, respectively).
6.75 2.23 0.76 1.57 0.53
+ + ± 4. 4-
0.86 ~' 0.32 0.11 0.[7 0.(12
279 (72°/,, of control) weeks. The livers of Aroclor 1254-treated rats were enlarged with fused lobes and liver weights were significantly increased at both time points, thus the liver/body weight ratio was increased significantly (Table I). In contrast, there were no significant changes in organ weights and organ/body weight ratios for kidneys and lungs.
Liver I-compounds Autoradiography showed (Fig. 1) three major I-spots on L maps (Nos. 1-3), four major spots on C maps (Nos. 4-7), and two major spots on U maps (Nos. 8 and 9) of corn oil-treated rat liver DNA. The unmarked spot close to the bottom edge of the U map (Fig. 1) and weaker unmarked spots on Figs. 1 and 4 were not quantified because of inconsistent intensities. The I-compound patterns were similar to those previously reported for male Sprague-Dawley rats [3,5,7,30]. Minor differences probably were a consequence of the different animal ages and diets. Two weeks after Aroclor 1254 treatment, the intensities of L spots 1-3, C spots 4-7, and U spots 8 and 9 (autoradiograms not shown) were reduced. R A L x 10 9 values for I-compound spots of L, C, and U maps and the total values are shown in Fig. 2. In the treated group L spot 1 was not detectable at 2 weeks, while reductions of L spots 2 and 3 and C spots 4 - 7 were significant (Fig. 2). The decreases in L and C total RALs were also significant at this time point (Fig. 2), while decreases in R A L values of U spots 8 and 9 were not significant (Fig. 2; P < 0.10). The combined total of L, C, and U maps was lowered significantly from 128.00 ± 21.89 to 73.30 4- 11.09 (P < 0.02). At 6 weeks, L spot 1 as well as C spots 6 and 7 were undetectable (autoradiograms not shown). The R A L values of spots 2 - 5 and 8 and 9 were significantly reduced at 6 weeks in Aroclor 1254-treated rat liver D N A compared to 6 weeks corn oil controls (Fig. 2). Decreases in total R A L values of L, C, and U maps were significant (Fig. 2) and similarly, the combined total L, C, and U values were reduced significantly from 134.10 4- 23.60 in the corn oil group to 47.83 4- 4.77 in the Aroclor 1254 group (P < 0.01).
Kidney I-compounds Autoradiography showed that kidney D N A from corn oil-treated rats gave similar I-spot patterns (Fig. 1) at 2 and 6 weeks, with four spots on L (Nos. 1-4), four spots on C (Nos. 5-8), and two spots on U (Nos. 9 and 10) maps. A comparison of the corn oil and Aroclor 1254 maps at 2 weeks showed intensities of L, C, and U spots not to be affected by the treatment (autoradiograms not shown). R A L values of kidney I-spots (Nos. 1-10) and L, C, and U totals (T) are shown in Fig. 3. The only significant change was an increase of the R A L of L spot 3 from 1.69 -4- 0.21 to 3.53 ± 0.36 (P < 0.001). The combined totals of L, C, and U regions remained unchanged. Comparison of L and C autoradiograms at 6 weeks (not shown) showed no treatment related changes in the L and U maps but intensities of C spots 5 and 6 decreased in the Aroclor 1254 group. R A L values of all individual I-spots (1-10) and totals are shown in Fig. 3. The decreases in R A L values of C spots 5 and 6 and C total were statistically significant. The combined totals of kidney R A L values from L, C,
dl
U
Zig. 1. Autoradiographic patterns of 32p-postlabeled l-compounds from male Sprague-Dawley rat liver and kidney DNA. Details of thin-layer chromaography (TLC) were as described [30]. Film exposure employed Kodak XAR-5 film with screen intensification at -80°C for 10-16 h. Locations of liver spots i-9 and kidney spots 1-10 have been indicated. L non-polar l-compounds from L cut-outs; C, moderately polar l-compounds from C cut-outs; U, polar -compounds from U cut-outs; B, background spot. For details see Materials and methods.
.IVER
KIDNEY
C
281 120I00o'1 o
80-
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60-
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Corn Oil Aroclor
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6420
n.1, n. I 1
2
2 WEEKS
3
n. , 1, I
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3
6 WEEKS
Fig. 2. Individual and total (T) R A L x 109 values (means ± S.D. from three individual rat livers) of L group (1-3, bottom panel), C group (4-7, middle panel), and U group (8 and 9, top panel) 1-compounds 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). All differences between treated and control groups (except spots 8 and 9 at 2 weeks) were significant (P < 0.05), and most P values were < 0.01 or < 0.001. *Indicates that the radioactivity of this spot was equal to or less than that of an adjacent blank area.
282 100 Corn Oil Aroclor
I
80 O3 0
60
X ..J
<
40 20-
0
9
II
10
1i
9
10
2 WEEKS
6 WEEKS
6050ol 0 X _J < rY
Corn Oil Aroclor
I
403020.
ni
10. 0
5
8 6 7 2 WEEKS
lI 5
li 6
7
8
6 WEEKS
60. r--I 50 (33 o
40
x .J < rY
30
I
Corn Oil Aroclor
20, 10
0
nl 1
tl ~,1
2 3 4 2 WEEKS
1
II nL 2
3
4
6 WEEKS
Fig. 3. Individual and total (T) R A L × 109 values (means 4- S.D. from three individual rat kidneys) or L group (1-4, bottom panel), C group (5-8, middle panel), and U group (9 and 10, top panel) I-spots, 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). Significant decreases in RALs were observed only for spots 5 and 6 and total (T) of C region at 6 weeks after Aroclor 1254 exposure (P < 0.001).
283 and U areas at 6 weeks (corn oil, 134.95 ± 6.15; Aroclor 1254, 138.88 ± 24.84) were not significantly affected by the treatment.
Lung 1-compounds Analysis of lung DNA from corn oil-treated control rats showed four spots on L (Nos. 1-4), two spots on C (Nos. 5, 6), and one spot on U (No. 7) maps (Fig. 4). Examination of corn oil and Aroclor 1254-treated DNA autoradiograms showed diminished intensities of L spots 1, 3, and 4 at 2 weeks. In the experimental group levels of L spots 1 and 3 were significantly reduced and spot 4 was undetectable (Fig. 5), while C and U spots were unaffected. The combined total RAL values of L, C, and U regions were not affected at 2 weeks. However, RAL values of all four L spots were decreased at 6 weeks (Fig. 5), while again C and U spots were not altered. The combined totals of L, C, and U maps were significantly decreased from 27.03 ± 1,35 to 21.75 ± 2.25 (P < 0.05). Thymidine incorporation In Aroclor 1254-treated rat liver DNA, [3H]TdR incorporation at 2 and 6 weeks was 3.7 and 6.3 times, respectively, that of corn oil controls (Table II). These increases were statistically significant (see Table II). There was a small but significant decrease in thymidine incorporation into kidney DNA of the Aroclor 1254 treated rats at 2 weeks only (Table II). Lung DNA from treated rats showed significantly increased thymidine incorporation at 2 and 6 weeks (1.5- and 5.6-fold, respectively; Table II). Absence of DNA adducts in Aroclor 1254-treated tissues The highly sensitive 32p-postlabeling assay [27,28,30], which is capable of detecting aromatic and bulky hydrophobic DNA adducts with a sensitivity range of one adduct in 109-1010 DNA nucleotides after nuclease P1 enrichment [28], did not reveal any extra spots indicative of Aroclor 1254-DNA adducts on L, C, or U maps of liver, kidney, or lung DNA from treated rats. Discussion As shown herein, two consecutive treatments with Aroclor 1254 led to significant decreases in liver and lung DNA I-spots at 2 and 6 weeks. This is the first time that this effect has been reported in a tissue other than liver. In contrast, only minor changes were seen in kidney DNA. Treatment with other non-mutagenic carcinogens and tumor promoters like peroxisome proliferators [30], choline-devoid diet [32], and T C D D [24] also leads to reduction of I-compound levels in the target organ [5-7,9]. Possible mechanisms underlying the latter effect include: (i) increased DNA synthesis; (ii) decreased formation of I-compounds; and (iii) increased I-compound removal from DNA. Regarding mechanism (i), I-compounds are presumably formed via the gradual interaction of DNA with electrophilic substances and metabolic intermediates of hormonal [2] or dietary [4] origin. Newly synthesized DNA devoid of such modifications may dilute the I-compound levels of pre-existin~ DNA [30]. The observation
n
3
1
285
25.
0
Corn Oil
I
Aroclor
zo-
7
7 2 WEEKS
6
6
6 WEEKS
T
5
4T
1
6
T
12 10 0 f-
6
X 6 4
0 12
3
2 WEEKS
2
3
4
T
6 WEEKS
Fig. 5. Individual and total (T) RAL x 10’ values (means + S.D. from three individual rat lungs) of L group (l-4, bottom panel), C group (5, 6, middle panel), and U group (7. top panel) I-spots. 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). Details are as in the legend of Fig. 2.
286 TABLE II COMPARISON OF [3H]THYMIDINE INCORPORATION (CPM/50 #g DNA, ± S.D.) IN LIVER, KIDNEY, AND LUNG DNA OF CORN OIL AND AROCLOR 1254-TREATED MALE SPRAGUE-DAWLEY RATS* 6 Weeks
Time point
2 Weeks
Treatment
Corn oil
Aroclor 1254
Corn oil
Aroclor 1254
601.0 4- 53 344.3 4- 78 413.3 ± 113
2235.3 + 593b 160.0 4- 61c 613.0 4- 56c
467.3 + 59 186.3 4- 44 118.0 ±32
2952.7 ± 195~' 221.6 + 80 666.0 ± 140b
Liver Kidney Lung
groups consisted of 3 animals. a'b'csignificantly different from corn oil controls (P < 0.001, 0.01, and 0.05, respectively).
*All
that reductions o f liver and lung l - c o m p o u n d levels were associated with increased thymidine incorporation lends support to this mechanism. Thus enhanced D N A synthesis following A r o c l o r 1254 exposure may have played a role in lowering 1c o m p o u n d levels. However, as discussed below, additional mechanisms were probably involved also. First, if the decrease o f I - c o m p o u n d s were solely due to a dilution effect one would expect an identical decrease for each liver or lung I-spot. However, liver L and C spots were more affected than were U spots (Fig. 2), while only L spots were decreased in lung D N A (Fig. 5). Similarly, previous reports showed that the extent of individual I - c o m p o u n d reduction by T C D D [7] and choline-devoid diet [5] is differential rather than uniform. Second, in mice treated with carbon tetrachloride, liver lc o m p o u n d s remain low long after thymidine incorporation has returned to control values [8]. Third, analysis o f Morris h e p a t o m a D N A showed that l - c o m p o u n d levels in slow growing, well differentiated tumors are reduced to a similar extent as those in fast growing, poorly differentiated hepatomas [13], arguing against increased D N A synthesis being the sole explanation of this phenomenon. As to mechanism (ii), A r o c i o r 1254 contains individual PCBs capable o f attaining a molecular geometry similar to that o f T C D D [14,33]. These substances can thus bind to the T C D D (Ah) receptor and induce cytochromes P-4501AI and P-4501A2 (P-450c and P-450d, respectively). The availability o f endogenous electrophiles which bind to D N A m a y have been reduced because o f the induction of a variety o f enzymes, resulting in decreased formation o f l-compounds. Treatment with c y t o c h r o m e P-450 inducers like phenobarbital, 3-methylcholanthrene, and pregnenolone 16c~-carbonitrile is reported to lower liver I - c o m p o u n d levels [34]. However, T C D D (Ah) receptor mediated mechanisms alone cannot explain the carcinogenicity and/or t u m o r p r o m o t i n g effects o f all PCBs capable o f occupying the receptor (see below). Regarding mechanism (iii), enzyme induction may conceivably lead to enhanced D N A repair and removal o f I-compounds. A r o c l o r 1254 pretreatment in vivo is k n o w n to enhance the D N A repair response to amino acid pyrolysate mutagens in primary cultures o f rat hepatocytes [35].
287 Lung is the only extrahepatic organ where a t u m o r p r o m o t i n g effect o f A r o c l o r 1254 has been demonstrated [20]. Metabolites o f PCBs are k n o w n to selectively accumulate in h u m a n [36] and rat [37] lung tissue which might explain respiratory toxicity and t u m o r promotion. Kidney I - c o m p o u n d s changed very little in response to A r o c l o r 1254 exposure. There are no reports showing initiating or p r o m o t i n g action o f A r o c l o r 1254 and other PCBs in rodent kidney [14,38]. Thus, A r o c l o r 1254 selectively decreased the I - c o m p o u n d levels in the two target organs, i.e. liver and lung. This suggests a possible correlation between the carcinogenic and t u m o r p r o m o t i n g effect o f PCBs and diminished I - c o m p o u n d levels in target organs. As mentioned above, A r o c l o r 1254 is a weak agonist/partial antagonist at the T C D D (Ah) receptor [25]. However, the t u m o r p r o m o t i n g action o f A r o c l o r 1254 is not weak, being demonstrable even after a single administration [19]. Even though T C D D and A r o c l o r 1254 have m a n y biologic and toxic properties in c o m m o n , there appears to be a clear difference in terms o f effects on liver I-compounds, as T C D D affected these D N A modifications only in female rats [6,7] while A r o c l o r 1254 decreased liver I - c o m p o u n d s in male rats (present study). It is interesting to note that whereas the hepatocarcinogenicity o f T C D D is female specific [23], A r o c l o r 1254 is k n o w n to be hepatocarcinogenic in male and female rats [18]. This would indicate that T C D D (Ah) receptor mediated mechanisms alone c a n n o t explain the carcinogenicity o f all polychlorinated aromatics. Information so far available suggests that I - c o m p o u n d s are closely linked to important events in the cell in some as yet unexplained manner. Regulatory processes appear to control formation and repair o f I - c o m p o u n d s to maintain them at an optimal level. Chronic impairment o f D N A functions potentially resulting from reduced I - c o m p o u n d levels could then play a role in the carcinogenic and t u m o r promoting effects o f A r o c l o r 1254 and other chemicals.
Acknowledgements This work was supported in part by N I H Grants R37 C A 32157 awarded by the National Cancer Institute and P42 ES 04917 awarded by the National Institute o f Environmental Health Sciences.
References 1 K. Randerath, M.V. Reddy and R.M. Disher, Age- and tissue related DNA modifications in untreated rats: Detection by 32P-postlabeling assay and possible significance for spontaneous tumor induction and aging. Carcinogenesis, 7 (1986) 1615. 2 K. Randerath, D. Li and E. Randerath, Age-related DNA modifications (i-compounds): modulation by physiological and pathological processes. Mutat. Res., 238 (1990) 245. 3 K. Randerath, J.G. Liehr, A. Gladek and E. Randerath, Age-dependent covalent DNA alterations (l-compounds) in rodent tissues: species, tissue and sex specificities. Mutat. Res., 219 (1989) 121. 4 D. Li and K. Randerath, Association between diet and age related DNA modifications (lcompounds) in rat liver and kidney. Cancer Res., 50 (1990) 3991. 5 D. Lk N. Chandar, B. Lombardi and K. Randerath, Reduced accumulation of l-compounds in liver DNA of rats fed a choline-devoid diet. Carcinogenesis, 10 (1989) 605.
288 6
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8 9
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11 12 13 14 15 16 17 18 19 20
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25
26 27
K. Randerath, K.L. Putman, E. Randerath, G. Mason, M. Kelley and S. Safe, Organ-specific effects of long term feeding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 1,2, 3,7,8-pentachlorodibenzo-pdioxin on 1-compounds in hepatic and renal DNA of female Sprague-Dawley rats. Carcinogenesis, 9 (1988) 2285. K. Randerath, K,L. Putman, E. Randerath, T. Zacharewski, M. Harris and S. Safe, Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on I-compounds in hepatic DNA of Sprague-Dawley rats: sex specific effects and structure-activity-relationships. Toxicol. Appl. Pharmacol., 103 (1990) 271. R.G. Nath, D. Li and K. Randerath, Acute and long-term effects of carbon tetrachloride on DNA modifications (I-compounds) in male mouse liver. Chem.-Biol. Interact., 76 (1990) 343. E. Randerath, K. Randerath, R. Reddy, T.F. Danna, M.S. Rao and J.K. Reddy, Induction of rat liver DNA alterations by chronic administration of peroxisome proliferators as detected by 32p_ postlabeling. Mutat. Res., 247 (1991) 65. E. Randerath, R.W. Hart, A. Turturro, T.F. Danna, R. Reddy and K. Randerath, Effects of aging and caloric restriction on 1-compounds in liver, kidney and white blood cell DNA of male BrownNorway rats. Mech. Aging Dev., 58 (1991) 279. V.N. Anisimov, Interaction between carcinogens and macromolecules and aging, in V.N. Anisimov (Ed.) Carcinogenesis and Aging, CRC Press Inc., Boca Raton, Florida, 1983, p. 111. D. Li and K. Randerath, Strain differences of I-compounds in relation to organ sites of spontaneous tumorigenesis and non-neoplastic renal diseases in mice. Carcinogenesis, 11 (1990) 251. K. Randerath, E. Randerath and T.F. Danna, Lack of I-compounds in DNA from a spectrum of Morris hepatomas. Carcinogenesis, 11 (1990) 1041. E.M. Silberhorn, H.P. Glauert and L.W. Robertson, Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol., 20 (1990) 440. D.P. Brown, Mortality of workers exposed to polychlorinated biphenyls - - An update. Arch. Environ. Health, 42 (1987) 333. R.S. Schoeny, C.C. Smith and J.C. Loper, Non-mutagenicity for Salmonella of the chlorinated hydrocarbons Aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone. Murat. Res., 68 (1979) 125. R.D. Kimbrough and R.E. Linder, Induction of adenofibrosis and hepatomas of the liver in BALB/cJ mice by polychlorinated biphenyls (Aroclor 1254). J. Natl. Cancer Inst., 53 (1974) 547. J.M. Ward, Proliferative lesions of the glandular stomach and liver in F344 rats fed diets containing Aroclor 1254. Environ. Health Perspect., 60 (1985)89. M.A. Pereira, S.L. Herren, A.L. Britt and M.M. Khoury, Promotion by polychlorinated biphenyls of enzyme-altered loci in rat liver. Cancer Lett., 15 (1982) 185. L.M. Anderson, J.M. Ward, S.D. Fox, H.J. Isaaq and C.W. Riggs, Effects of a single dose of polychlorinated biphenyls to infant mice on N-nitrosodimethylamine-initiatedlung and liver tumors. Int. J. Cancer, 38 (1986) 109. L.M. Anderson, S.S. Hecht, D.E. Dixon, L.F. Dove, R.M. Kovatch, S, Amin, D. Hoffmann and J.M. Rice, Evaluation of the transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-I-butanonein mice. Cancer Res., 49 (1989) 3770. B.D. Preston, J.P. Van Miller, R.W. Moore and J.R. Allen, Promoting effects of polychlorinated biphenyls (Aroclor 1254) and polychlorinated dibenzofuran-free Aroclor 1254 on diethylnitrosamine-induced tumorigenesis in the rat. J. Natl. Cancer Inst., 66 (1981) 509. R.J. Kociba, D.J. Keyes, J.E. Beyer, R.M. Carreon, C.E. Wade, D.A. Dittenber, R.P. Kalnins, L.E. Frauson, C.N. Park, S.D. Barnard, R.A. Hummel and C.G. Humiston, Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol., 46 (1978) 279. H.C. Pitot, T. Goldsworthy, H.A. Campbell and H. Poland, Quantitative evaluation of the promotion of 2,3,7,8-tetrachlorodibenzo-p-dioxinof hepatocarcinogenesis from dimethylnitrosamine. Cancer Res., 40 (1980) 3616. R. Bannister, D. Davis, T. Zacharewski, 1. Tizard and S. Safe, Aroclor 1254 as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: effects on enzyme induction and immunotoxicity. Toxicology, 46 (1987) 29. R.C. Gupta, M.V. Reddy and K. Randerath, 32p-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis, 3 (1982) 1081. M.V. Reddy, R.C. Gupta, E. Randerath and K. Randerath, 32p-postlabeling test for covalent DNA
289
28 29 30
31 32 33 34 35 36 37
38
binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents. Carcinogenesis, 5 (1984) 231. M.V. Reddy and K. Randerath, Nuclease Pl-mediated enhancement of sensitivity of 32p_ postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7 (1986) 1543. R.C. Gupta, Non-random binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. USA, 81 (1984) 6943. K. Randerath, L.-J.W. Lu and D. Li, A comparison between different types of covalent DNA modifications (I-compounds, persistent carcinogen adducts and 5-methylcytosine) in regenerating rat liver. Carcinogenesis, 9 (1988) 1843. J.K. Reddy and M.S. Rao, Enhancement by Wy-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine initiated hepatic tumorigenesis in the rat. Br. J. Cancer, 40 (1979) 476. S. Yokoyama, M.A. Sell, T.V. Reddy and B. Lombardi, Hepatocarcinogenic and promoting action of a choline-devoid diet i,1 the rat. Cancer Res., 45 (1985) 2834. S. Safe, Polychlorinated biphenyls (PCBs): mutagenicity and carcinogenicity. Mutat. Res., 220 (1989) 31. D. Li, S. Chen, B. Moorthy and K. Randerath, Modulation of I-compounds by cytochrome P-450 inducers in rat liver DNA. Proc. Am. Assoc. Cancer Res., 32 (1991) 108. D.J. Loury and J.L. Byard, Aroclor 1254 pretreatment enhances the DNA repair response to amino acid pyrolysate mutagens in primary cultures of rat hepatocytes. Cancer Lett., 20 (1983) 283. H. Haraguchi, H. Kuroki and Y. Masuda, Determination of methylthio and methylsulphone polychlorinated biphenyls in tissues of patients with 'Yusho'. Food Chem. Toxicol., 22 (1984) 283. J. Lund, L. Nordlund and J.A. Gustafsson, Partial purification of a binding protein for polychlorinated biphenyls from rat lung cytosol: physicochemical and immunochemical characterization. Biochem., 27 (1988) 7895. M. Hirose, T. Shirai, H. Tsuda, S. Fukushima, T. Ogiso and N. lto, Effect of phenobarbital, polychlorinated biphenyl and sodium saccharin on hepatic and renal carcinogenesis in unilaterally nephrectomized rats given N-ethyl-N-hydroxyethylnitrosamine orally. Carcinogenesis, 2 (1981) 1299.
275
Short-term effects of the tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on I-compounds in liver, kidney and lung DNA of male Sprague-Dawley rats Raghu G. Nath, Erika Randerath and Kurt Randerath Division of Toxicology. Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) (Received March 27tb, 1991; accepted June 12th, 1991)
Summary The effects of a tumor promoting polychlorinated biphenyl mixture, Aroclor 1254, on l-compounds (tissue, species and sex dependent D N A modifications that increase with age in untreated rodents) were studied by 32p-postlabeliffg in male Sprague-Dawley rat liver, kidney, and lung DNA. Aroclor 1254 was dissolved in corn oil and intraperitoneally (i.p.) injected (2 x 500 mg/kg, 2 weeks apart) into 3-month-old rats. Control rats were given corn oil. Groups of 3 animals were sacrificed at 2 and 6 weeks after the second injection of corn oil or Aroclor 1254. At both time points Aroclor 1254-treated rats had significantly lower body weights and higher liver weights while kidney and lung weights were unaffected. Thymidine incorporation into liver and lung D N A was significantly increased at both time points, while kidney D N A showed a small decrease at 2 weeks. Treatment resulted in significant reductions (ranging from 29 to 100'%) of each of nine liver I-spots at 2 and 6 weeks. In treated rats there was no decrease in kidney I-spots at 2 weeks, while the levels of only two out of ten kidney spots were reduced by 42-91'7o at 6 weeks. At 2 weeks three out of seven and at 6 weeks four out of seven lung I-spots were lowered by 51-100% in the Aroclor 1254-treated rats. Thus the effects decreased in the order liver > lung > kidney. Since Aroclor 1254 has been reported to be a tumor promoter in liver and lung but not kidney, these results suggest a correlation between organ specific promotion of carcinogenesis by Aroclor 1254 and the reduction of D N A l-compounds.
Key words: Aroclor 1254; Carcinogenesis; D N A synthesis, I-compounds: Polychlorinated biphenyls; T u m o r promotion
Correspondence to: Kurt Randerath, Division of Toxicology, Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, U.S.A. Abbreviations: Ah, aryl hydrocarbon; C, central; D, direction; [3H]TdR, [methyl-3H]thymidine; Icompounds, D N A modifications which tend to increase with age in tissues of untreated animals: L, lower; PCB, polychlorinated biphenyl; PEI, polyethyleneimine; RAL, relative adduct labeling, a measure for the level of covalent modifications of DNA; T C D D , 2,3,7,8-tetrachlorodibenzo-p-dioxin: TLC, thin-layer chromatography; U, upper. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
276 Introduction
Novel covalent DNA modifications termed I(indigenous)-compounds have been detected by the 32p-postlabeling assay for DNA adducts in various tissues of untreated rodents [1,2]. I-compounds tend to gradually accumulate with increasing animal age, and their profiles and levels depend on species, sex, tissue, and diet [2,3]. I-compounds are presumably formed by the interaction of DNA with endogenous electrophilic metabolites of hormonal [2] or dietary [4] origin. Procedures known to induce liver cancer, i.e. feeding a choline devoid diet [5] or treatment with nonmutagenic hepatocarcinogens such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [6,7], carbon tetrachloride [8], and peroxisome proliferators [9] result in significantly diminished I-compound levels in rodent liver. Rat liver I-compound levels reach a plateau at about 16-24 months of age, remaining unchanged for the most part as the animals grow older [10] and become more susceptible to spontaneous tumorigenesis [11]. Strains of mice highly susceptible to spontaneous hepatomas have relatively lower levels of liver l-compounds than less susceptible strains [12]. I-compounds were found virtually absent from a spectrum of Morris hepatomas [13]. Thus a multitude of observations suggests that reduced I-compound levels are associated with, and may perhaps contribute to carcinogenesis. Polychlorinated biphenyls (PCBs) are chemically stable environmental contaminants now widely distributed in every component of the global ecosystem. The toxic effects of PCBs include thymic atrophy, a wasting syndrome, immunotoxic responses, hepatotoxicity, and chloracne [14]. Available evidence does not conclusively show a greater cancer risk in humans exposed to PCBs [15]. Since highly chlorinated PCBs are poorly metabolized in the body they are not readily activated to reactive metabolites capable of covalent binding to DNA and tissue macromolecules. As a result they are likely to act as tumor promoters rather than initiators. Aroclor 1254 is inactive in mutagenicity tests using the Ames Salmonella assay [16]. Chronic feeding of high doses of Aroclor 1254 induces liver tumors in mice [17] and rats [18]. The tumor promoting effects of PCBs are studied mostly in Sprague-Dawley and F344 rats as well as various strains of mice. Aroclor 1254, Kaneclor 500, and other PCB mixtures (unspecified components) are commonly employed in promotion studies [14]. Studies using the two-stage hepatocarcinogenesis model clearly indicate the tumor promoting effect of Aroclor 1254. In partially hepatectomized male SpragueDawley rats given diethylnitrosamine, one dose of Aroclor 1254 increases the incidence of gamma-glutamyltranspeptidase-positive foci [19], while in rats not treated with diethylnitrosamine, Aroclor 1254 treatment does not increase the incidence of such foci indicating the lack of an initiating effect. In mice initiated with dimethylnitrosamine, Aroclor 1254 increases the size of hepatic tumors and foci and also the incidence of lung tumors [20]. In another study [21], when the tobaccospecific carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone was administered to pregnant mice and the progeny were given a single dose of Aroclor 1254 the incidence of hepatic tumors significantly increased. The tumor promoting effect of Aroclor 1254 in male Sprague-Dawley rat liver was shown not to be due to polychlorinated dibenzofuran impurities [22].
277 Some of the biologic and toxic effects of PCBs resemble those exhibited by polychlorinated dibenzo-p-dioxins. Previous reports showed T C D D to be a hepatocarcinogen in female rats [23] and also a tumor promoter [24]. Reports from our laboratory have shown that T C D D selectively decreases hepatic I-compound levels in the female rat [6,7]. T C D D is not carcinogenic to the kidney [23] and the kidney I-compound level is not affected in the female rat [6,7]. Aroclor 1254, like TCDD, is known to competitively bind to the specific aryl hydrocarbon (Ah) receptor [25]. In the present study we investigated the effect of Aroclor 1254 on liver, kidney, and lung I-compounds in male Sprague-Dawley rats. Materials and methods
Materials [methyl-3H]thymidine ([3H]TdR, 71 Ci/mmol in 70% ethanol) and aqueous [32p]phosphate (HCI-free; >300 mCi/ml) were purchased from ICN Radiochemicals (Irvine, CA). The sources of all other biochemical and chromatographic materials used for adduct analysis have been previously reported [26-28]. Animals and treatment Male Sprague-Dawley rats (3 months old, 230-260 g) were purchased from Harlan Sprague-Dawley, Inc. (Houston, TX). The rats were housed three per cage and fed Wayne Lab Chow ( M R H 22/5 Rodent Blox) and tap water ad libitum. Aroclor 1254 (Analabs, New Haven, CT) was injected intraperitoneally (i.p.) at a dose of 500 mg/kg. To administer this dose the compound was dissolved in corn oil (Sigma, St. Louis, MO) to make a 400 mg/ml solution and i.p. injected in a volume of 0.125 ml/100 g body weight. Twelve rats were divided into four groups of three animals each. Rats of groups 1 and 3 (control) received two injections of corn oil (0.125 ml/100 g) 2 weeks apart and the rats of groups 2 and 4 (experimental) were given two injections of Aroclor 1254 (500 mg/kg) 2 weeks apart. Two weeks after the second injection, rats of groups 1 and 2 were given three i.p. injections each of [3H]TdR (150/~Ci/kg) every 2 h starting at 1000 h. The rats were sacrificed 2 h after the third dose of [3H]TdR, the liver, kidneys, and lungs were removed, cleaned, weighed, minced, and frozen at -80°C until DNA isolation. Six weeks after the second dose of corn oil or Aroclor 1254, rats of groups 3 and 4 were sacrificed and tissues removed after similar [3H]TdR treatments. DNA isolation and 32p-postlabeling DNA was isolated from liver, kidneys, and lungs of three individual animals by solvent extraction combined with enzymatic digestion of proteins and RNA [29]. The amount of radioactivity (3H) incorporated into DNA was determined by counting 50 #g DNA from each individual organ in a Packard Scintillation Counter (Tricarb 4000 Series) using 10 ml Aqueous Counting Scintiilant (ACS, Amersham Corporation, Arlington Heights,lL). DNA from each sample was analyzed in duplicate by a nuclease PI enhanced 32p-postlabeling assay [28]. Briefly, 10 /~g DNA was hydrolyzed to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase, followed by
278 incubation with nuclease P1 to 3'-dephosphorylate normal nucleotides [28]. Digestion was terminated by the addition of 3.0 /~1 of 2-[N-cyclohexylamino]-ethane sulfonic acid (CHES) buffer (0.5 M, pH 9.6), then the mixture was evaporated to dryness and reconstituted in 10 t~l distilled water. The adducts were labeled with excess carrier-free [3,-32p]ATP (3500-5000 Ci/mmol), spotted on a polyethylene±mine (PEI)-cellulose thin layer and developed overnight in 2.3 M sodium phosphate, pH 5.75 (D1) to remove excess ATP and normal nucleotides [27,28]. The non-polar Icompounds retained in the origin area (L, lower cut) and more polar I-compounds which migrated out of the origin [center (C) and upper (U) cuts] were contacttransferred to three separate fresh PEI-cellulose sheets and resolved by twodimensional thin-layer chromatography (TLC) as previously described [30]. Age dependent increases were not detected for the majority of I-compounds, probably because the 4 week observation period was too short. 32p-labeled fractions were identified as I-compounds by TLC comparison with previously characterized I-compounds in male rats [3,5,7,30]. 32p-labeled l-spots were located by screenenhanced autoradiography, excised, and subjected to scintillation (Cerenkov) assay for estimation of relative adduct labeling (RAL) values [30]. These values were calculated from the net count rates of spots, the specific activity of ATP, and the amount of DNA as described [28,30] and were expressed as RAL x 109 values, representing a minimum estimate of the number of modified nucleotides in 109 DNA nucleotides. Means ± standard deviations (S.D.) of RAL values were evaluated for significant differences by Student's t-test. Results
Tissue and body weights Table I compares the mean ( + S.D.) body weights, organ weights, and organ/body weight ratios from corn oil and Aroclor 1254-treated rats. Treated rats had significantly lower body weights than control rats at 2 (76% of control) and 6
TABLE 1 EFFECT
OF AROCLOR
SPRAGUE-DAWLEY
1254 O N B O D Y , L I V E R , K I D N E Y ,
AND LUNG WEIGHTS
IN M A L E
RATS* 6 Weeks
Time point
2 Weeks
Treatment
C o r n oil
A r o c l o r 1254
C o r n oil
A r o c l o r 1254
356.30 4- 12.60 12.91 ± 1.42
271.30 ± 8.50 a 17.85 ± 1.34 b
408.30 ± 28.00 11.85 ± 1.05
294.30 .4- 25.70 a 19.87 4. 1.35 b
B o d y wt (g) Liver wt (g) L i v e r / B o d y wt (%) K i d n e y wt (g) K i d n e y / B o d y wt (%) L u n g wt (g)
3.62 2.61 0.73 1.53
± 4± 4-
0.44 0.15 0.03 0.09
L u n g / B o d y w t (%)
0.43 ± 0.02
6.58 2.27 0.84 1.70
44:t: 4-
0.46 b 0.29 0.08 0.43
0.63 4- 0,16
2.90 2.48 0.61 1.90 0.47
+ + ± .44-
0.06 0.21 0.01 0.65 0.13
*All g r o u p s c o n s i s t e d o f 3 a n i m a l s . a,bSignificantly different f r o m c o r n oil c o n t r o l s ( P < 0.001 a n d 0.01, respectively).
6.75 2.23 0.76 1.57 0.53
+ + ± 4. 4-
0.86 ~' 0.32 0.11 0.[7 0.(12
279 (72°/,, of control) weeks. The livers of Aroclor 1254-treated rats were enlarged with fused lobes and liver weights were significantly increased at both time points, thus the liver/body weight ratio was increased significantly (Table I). In contrast, there were no significant changes in organ weights and organ/body weight ratios for kidneys and lungs.
Liver I-compounds Autoradiography showed (Fig. 1) three major I-spots on L maps (Nos. 1-3), four major spots on C maps (Nos. 4-7), and two major spots on U maps (Nos. 8 and 9) of corn oil-treated rat liver DNA. The unmarked spot close to the bottom edge of the U map (Fig. 1) and weaker unmarked spots on Figs. 1 and 4 were not quantified because of inconsistent intensities. The I-compound patterns were similar to those previously reported for male Sprague-Dawley rats [3,5,7,30]. Minor differences probably were a consequence of the different animal ages and diets. Two weeks after Aroclor 1254 treatment, the intensities of L spots 1-3, C spots 4-7, and U spots 8 and 9 (autoradiograms not shown) were reduced. R A L x 10 9 values for I-compound spots of L, C, and U maps and the total values are shown in Fig. 2. In the treated group L spot 1 was not detectable at 2 weeks, while reductions of L spots 2 and 3 and C spots 4 - 7 were significant (Fig. 2). The decreases in L and C total RALs were also significant at this time point (Fig. 2), while decreases in R A L values of U spots 8 and 9 were not significant (Fig. 2; P < 0.10). The combined total of L, C, and U maps was lowered significantly from 128.00 ± 21.89 to 73.30 4- 11.09 (P < 0.02). At 6 weeks, L spot 1 as well as C spots 6 and 7 were undetectable (autoradiograms not shown). The R A L values of spots 2 - 5 and 8 and 9 were significantly reduced at 6 weeks in Aroclor 1254-treated rat liver D N A compared to 6 weeks corn oil controls (Fig. 2). Decreases in total R A L values of L, C, and U maps were significant (Fig. 2) and similarly, the combined total L, C, and U values were reduced significantly from 134.10 4- 23.60 in the corn oil group to 47.83 4- 4.77 in the Aroclor 1254 group (P < 0.01).
Kidney I-compounds Autoradiography showed that kidney D N A from corn oil-treated rats gave similar I-spot patterns (Fig. 1) at 2 and 6 weeks, with four spots on L (Nos. 1-4), four spots on C (Nos. 5-8), and two spots on U (Nos. 9 and 10) maps. A comparison of the corn oil and Aroclor 1254 maps at 2 weeks showed intensities of L, C, and U spots not to be affected by the treatment (autoradiograms not shown). R A L values of kidney I-spots (Nos. 1-10) and L, C, and U totals (T) are shown in Fig. 3. The only significant change was an increase of the R A L of L spot 3 from 1.69 -4- 0.21 to 3.53 ± 0.36 (P < 0.001). The combined totals of L, C, and U regions remained unchanged. Comparison of L and C autoradiograms at 6 weeks (not shown) showed no treatment related changes in the L and U maps but intensities of C spots 5 and 6 decreased in the Aroclor 1254 group. R A L values of all individual I-spots (1-10) and totals are shown in Fig. 3. The decreases in R A L values of C spots 5 and 6 and C total were statistically significant. The combined totals of kidney R A L values from L, C,
dl
U
Zig. 1. Autoradiographic patterns of 32p-postlabeled l-compounds from male Sprague-Dawley rat liver and kidney DNA. Details of thin-layer chromaography (TLC) were as described [30]. Film exposure employed Kodak XAR-5 film with screen intensification at -80°C for 10-16 h. Locations of liver spots i-9 and kidney spots 1-10 have been indicated. L non-polar l-compounds from L cut-outs; C, moderately polar l-compounds from C cut-outs; U, polar -compounds from U cut-outs; B, background spot. For details see Materials and methods.
.IVER
KIDNEY
C
281 120I00o'1 o
80-
x
60-
IHI
Corn Oil Aroclor
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.< tY
40200
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9
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6420
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Fig. 2. Individual and total (T) R A L x 109 values (means ± S.D. from three individual rat livers) of L group (1-3, bottom panel), C group (4-7, middle panel), and U group (8 and 9, top panel) 1-compounds 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). All differences between treated and control groups (except spots 8 and 9 at 2 weeks) were significant (P < 0.05), and most P values were < 0.01 or < 0.001. *Indicates that the radioactivity of this spot was equal to or less than that of an adjacent blank area.
282 100 Corn Oil Aroclor
I
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60
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<
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0
9
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Fig. 3. Individual and total (T) R A L × 109 values (means 4- S.D. from three individual rat kidneys) or L group (1-4, bottom panel), C group (5-8, middle panel), and U group (9 and 10, top panel) I-spots, 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). Significant decreases in RALs were observed only for spots 5 and 6 and total (T) of C region at 6 weeks after Aroclor 1254 exposure (P < 0.001).
283 and U areas at 6 weeks (corn oil, 134.95 ± 6.15; Aroclor 1254, 138.88 ± 24.84) were not significantly affected by the treatment.
Lung 1-compounds Analysis of lung DNA from corn oil-treated control rats showed four spots on L (Nos. 1-4), two spots on C (Nos. 5, 6), and one spot on U (No. 7) maps (Fig. 4). Examination of corn oil and Aroclor 1254-treated DNA autoradiograms showed diminished intensities of L spots 1, 3, and 4 at 2 weeks. In the experimental group levels of L spots 1 and 3 were significantly reduced and spot 4 was undetectable (Fig. 5), while C and U spots were unaffected. The combined total RAL values of L, C, and U regions were not affected at 2 weeks. However, RAL values of all four L spots were decreased at 6 weeks (Fig. 5), while again C and U spots were not altered. The combined totals of L, C, and U maps were significantly decreased from 27.03 ± 1,35 to 21.75 ± 2.25 (P < 0.05). Thymidine incorporation In Aroclor 1254-treated rat liver DNA, [3H]TdR incorporation at 2 and 6 weeks was 3.7 and 6.3 times, respectively, that of corn oil controls (Table II). These increases were statistically significant (see Table II). There was a small but significant decrease in thymidine incorporation into kidney DNA of the Aroclor 1254 treated rats at 2 weeks only (Table II). Lung DNA from treated rats showed significantly increased thymidine incorporation at 2 and 6 weeks (1.5- and 5.6-fold, respectively; Table II). Absence of DNA adducts in Aroclor 1254-treated tissues The highly sensitive 32p-postlabeling assay [27,28,30], which is capable of detecting aromatic and bulky hydrophobic DNA adducts with a sensitivity range of one adduct in 109-1010 DNA nucleotides after nuclease P1 enrichment [28], did not reveal any extra spots indicative of Aroclor 1254-DNA adducts on L, C, or U maps of liver, kidney, or lung DNA from treated rats. Discussion As shown herein, two consecutive treatments with Aroclor 1254 led to significant decreases in liver and lung DNA I-spots at 2 and 6 weeks. This is the first time that this effect has been reported in a tissue other than liver. In contrast, only minor changes were seen in kidney DNA. Treatment with other non-mutagenic carcinogens and tumor promoters like peroxisome proliferators [30], choline-devoid diet [32], and T C D D [24] also leads to reduction of I-compound levels in the target organ [5-7,9]. Possible mechanisms underlying the latter effect include: (i) increased DNA synthesis; (ii) decreased formation of I-compounds; and (iii) increased I-compound removal from DNA. Regarding mechanism (i), I-compounds are presumably formed via the gradual interaction of DNA with electrophilic substances and metabolic intermediates of hormonal [2] or dietary [4] origin. Newly synthesized DNA devoid of such modifications may dilute the I-compound levels of pre-existin~ DNA [30]. The observation
n
3
1
285
25.
0
Corn Oil
I
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7 2 WEEKS
6
6
6 WEEKS
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6
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6
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T
6 WEEKS
Fig. 5. Individual and total (T) RAL x 10’ values (means + S.D. from three individual rat lungs) of L group (l-4, bottom panel), C group (5, 6, middle panel), and U group (7. top panel) I-spots. 2 and 6 weeks after two i.p. injections of corn oil or Aroclor 1254 (see Materials and methods). Details are as in the legend of Fig. 2.
286 TABLE II COMPARISON OF [3H]THYMIDINE INCORPORATION (CPM/50 #g DNA, ± S.D.) IN LIVER, KIDNEY, AND LUNG DNA OF CORN OIL AND AROCLOR 1254-TREATED MALE SPRAGUE-DAWLEY RATS* 6 Weeks
Time point
2 Weeks
Treatment
Corn oil
Aroclor 1254
Corn oil
Aroclor 1254
601.0 4- 53 344.3 4- 78 413.3 ± 113
2235.3 + 593b 160.0 4- 61c 613.0 4- 56c
467.3 + 59 186.3 4- 44 118.0 ±32
2952.7 ± 195~' 221.6 + 80 666.0 ± 140b
Liver Kidney Lung
groups consisted of 3 animals. a'b'csignificantly different from corn oil controls (P < 0.001, 0.01, and 0.05, respectively).
*All
that reductions o f liver and lung l - c o m p o u n d levels were associated with increased thymidine incorporation lends support to this mechanism. Thus enhanced D N A synthesis following A r o c l o r 1254 exposure may have played a role in lowering 1c o m p o u n d levels. However, as discussed below, additional mechanisms were probably involved also. First, if the decrease o f I - c o m p o u n d s were solely due to a dilution effect one would expect an identical decrease for each liver or lung I-spot. However, liver L and C spots were more affected than were U spots (Fig. 2), while only L spots were decreased in lung D N A (Fig. 5). Similarly, previous reports showed that the extent of individual I - c o m p o u n d reduction by T C D D [7] and choline-devoid diet [5] is differential rather than uniform. Second, in mice treated with carbon tetrachloride, liver lc o m p o u n d s remain low long after thymidine incorporation has returned to control values [8]. Third, analysis o f Morris h e p a t o m a D N A showed that l - c o m p o u n d levels in slow growing, well differentiated tumors are reduced to a similar extent as those in fast growing, poorly differentiated hepatomas [13], arguing against increased D N A synthesis being the sole explanation of this phenomenon. As to mechanism (ii), A r o c i o r 1254 contains individual PCBs capable o f attaining a molecular geometry similar to that o f T C D D [14,33]. These substances can thus bind to the T C D D (Ah) receptor and induce cytochromes P-4501AI and P-4501A2 (P-450c and P-450d, respectively). The availability o f endogenous electrophiles which bind to D N A m a y have been reduced because o f the induction of a variety o f enzymes, resulting in decreased formation o f l-compounds. Treatment with c y t o c h r o m e P-450 inducers like phenobarbital, 3-methylcholanthrene, and pregnenolone 16c~-carbonitrile is reported to lower liver I - c o m p o u n d levels [34]. However, T C D D (Ah) receptor mediated mechanisms alone cannot explain the carcinogenicity and/or t u m o r p r o m o t i n g effects o f all PCBs capable o f occupying the receptor (see below). Regarding mechanism (iii), enzyme induction may conceivably lead to enhanced D N A repair and removal o f I-compounds. A r o c l o r 1254 pretreatment in vivo is k n o w n to enhance the D N A repair response to amino acid pyrolysate mutagens in primary cultures o f rat hepatocytes [35].
287 Lung is the only extrahepatic organ where a t u m o r p r o m o t i n g effect o f A r o c l o r 1254 has been demonstrated [20]. Metabolites o f PCBs are k n o w n to selectively accumulate in h u m a n [36] and rat [37] lung tissue which might explain respiratory toxicity and t u m o r promotion. Kidney I - c o m p o u n d s changed very little in response to A r o c l o r 1254 exposure. There are no reports showing initiating or p r o m o t i n g action o f A r o c l o r 1254 and other PCBs in rodent kidney [14,38]. Thus, A r o c l o r 1254 selectively decreased the I - c o m p o u n d levels in the two target organs, i.e. liver and lung. This suggests a possible correlation between the carcinogenic and t u m o r p r o m o t i n g effect o f PCBs and diminished I - c o m p o u n d levels in target organs. As mentioned above, A r o c l o r 1254 is a weak agonist/partial antagonist at the T C D D (Ah) receptor [25]. However, the t u m o r p r o m o t i n g action o f A r o c l o r 1254 is not weak, being demonstrable even after a single administration [19]. Even though T C D D and A r o c l o r 1254 have m a n y biologic and toxic properties in c o m m o n , there appears to be a clear difference in terms o f effects on liver I-compounds, as T C D D affected these D N A modifications only in female rats [6,7] while A r o c l o r 1254 decreased liver I - c o m p o u n d s in male rats (present study). It is interesting to note that whereas the hepatocarcinogenicity o f T C D D is female specific [23], A r o c l o r 1254 is k n o w n to be hepatocarcinogenic in male and female rats [18]. This would indicate that T C D D (Ah) receptor mediated mechanisms alone c a n n o t explain the carcinogenicity o f all polychlorinated aromatics. Information so far available suggests that I - c o m p o u n d s are closely linked to important events in the cell in some as yet unexplained manner. Regulatory processes appear to control formation and repair o f I - c o m p o u n d s to maintain them at an optimal level. Chronic impairment o f D N A functions potentially resulting from reduced I - c o m p o u n d levels could then play a role in the carcinogenic and t u m o r promoting effects o f A r o c l o r 1254 and other chemicals.
Acknowledgements This work was supported in part by N I H Grants R37 C A 32157 awarded by the National Cancer Institute and P42 ES 04917 awarded by the National Institute o f Environmental Health Sciences.
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