The pharmacokinetics of PCB 77 and PCB 180 in plasma and brain were similar with small but notable differences in a dose-dependent manner (Supplemental files 1A and 1B). These parameters could be correlated to central behavioral effects based on brain pharmacokinetics, and autonomic nervous system effects based on plasma pharmacokinetics, in dose-dependent and time-dependent manner (Supplemental files 3A and 3B). Generally, brain accumulation of PCB 77 was more significantly responsible for behavioral disruption than PCB 180 accumulation in plasma – based on brain-plasma ratios of both these congeners (Supplemental files 2A and 2B).
Pharmacokinetics profile of PCB 77 at 2.0, 4.0 and 16.0 mg.m-3 in mice plasma:
The mean maximum concentration attained, Cmax of PCB 77 in plasma was 88.32 ± 8.28 ng.ml-1 for the 2 mg.m-3 group. The time taken to achieve this value of Cmax in the plasma of low dose exposure group was 3.50 ± 1.00 hr. The AUC0-t for PCB 77 in plasma was 1321.19 ± 55.72 ng.hr.ml-1. The Area Under the plasma Concentration-time (infinity) Curve, AUC0-∞ in plasma was 1626.02 ± 64.13 ng.hr.ml-1 for PCB 77 after dosing 2 mg.m-3 of Aroclor 1232. The elimination rate constant, Kel in plasma for PCB 77 after dosing mice with 2 mg.m-3 of Aroclor 1232 by inhalation route was 0.03 hr-1. Plasma half-life of PCB 77 in this exposure group was 20.17 ± 1.38 hr. The Mean plasma Residence Times (MRT) for PCB 77 in this dose level was 16.51 ± 0.42 hr. The plasma apparent volume of distribution (Vd) and clearance (CL) for PCB 77 after dosing Aroclor 1232 in this group of mice were 35858.25 ± 3073.55 and, 1231.46 ± 49.43 ml.hr-1.m-2, respectively.
In the 4 mg.m-3 exposure group, the Cmax and Tmax of PCB 77 in plasma were 245.62 ± 39.58 ng.ml-1 and 4.00 hr, respectively. The AUC0-t and AUC0-∞ of PCB 77 in this intermediate dosing group were 2749.28 ± 392.00 and 2957.28 ± 413.20 ng.hr.ml-1 in plasma. The Kel values were 0.06 hr-1 in plasma of the mice treated with 4 mg.m-3 group, while the half-life was 12.72 ± 0.36 hr in this group. The MRT in the intermediate dose exposure groups was 14.31 ± 0.49 hr in the plasma. Finally, the Vd and CL in plasma of this exposure level were 25209.01 ± 3835.06 ml.m-2 and 1375.07 ± 215.15 ml.hr-1.m-2.
In the 16 mg.m-3 group, the Cmax of PCB 77 was 727.40 ± 42.52 ng.ml-1 in plasma and Tmax was 4.00 hr. The mean AUC0-t and AUC0-∞ in the plasma of this high dose group were 7565.21 ± 148.44 and 8013.39 ± 263.11 ng.hr.ml-1. In the high dose group, the Keland t-1/2 in plasma were 0.06 hr-1, and 11.17 ± 1.54 hr, respectively. The MRT of PCB 77 in plasma of the mice treated with high dose of Aroclor 1232was 14.29 ± 0.24 hr. The high dose group Vd and CL in plasma was 32099.78 ± 3593.04 ml.m-2 and 1998.28 ± 65.96 ml.hr-1.m-2 in plasma.
The mean plasma concentration-time profile of PCB 77 in mouse plasma after inhalation of PCB mixture at three different dose levels are presented in Figure 1, inset A, and Table 1.
Pharmacokinetics profile of PCB 77 at 2.0, 4.0 and 16.0 mg.m-3 in mice brain:
The mean maximum concentration attained; Cmax of PCB 77 was 69.43 ± 10.54 ng.ml-1 in brains of mice treated with 2 mg.m-3 of Aroclor 1232 via inhalation route. The Tmax of PCB 77 in brain tissues of mice exposed to this dose was 3.50 ± 1.00 hr. The AUC0-t for PCB 77 in brain was 1254.97 ± 57.51 ng.hr.ml-1. The AUC0-∞ was 1469.62 ± 161.80 ng.hr.ml-1 for PCB 77. The elimination rate constant, Kel in brain for PCB 77 was 0.05 hr-1. Brain half-life of PCB 77 in this exposure group was 16.20 ± 4.36 hr. The Mean brain Residence Times (MRT) for PCB 77 in this dose level was 17.07 ± 1.93 hr. The values for the apparent volume of distribution and tissue clearance of PCB 77 in brain were 31481.64 ± 5927.66 ml.m-2 respectively, and 1373.32 ± 150.80 ml.hr-1.m-2, respectively.
In the 4 mg.m-3 exposure group, the Cmax and Tmax of PCB 77 in brain were 175.13 ± 6.42 ng.ml-1, and 4.00 hr, respectively. The AUC0-t and AUC0-∞ of PCB 77 in this group were 2581.12 ± 136.46 and 2366.40 ± 142.19 ng.hr.ml-1, respectively. The Kel values were 0.05 hr-1 in brain of the mice treated with 4 mg.m-3 group, while the half-lifeof PCB 77 in brain was 14.20 ± 0.58 hr in brain. The MRT in the intermediate dose exposure group was 15.05 ± 0.32 hr in brain, while the Vd and CL in brain of this exposure group were 31856.13 ± 3012. 73 ml.m-2 and 1553.12 ± 85.97 ml.hr-1.m-2.
In the 16 mg.m-3 group, the Cmax of PCB 77 was 526.21 ± 28.63 ng.ml-1 in brain and Tmax was 4.00 hr. The mean AUC0-t and AUC0-∞ in the brain of this group were 6798.93 ± 340.47 ng.hr.ml-1and 7100.56 ± 413.77ng.hr.ml-1. The Kel and t-1/2 in brain were 0.07 hr-1, and 10.82 ± 1.29 hr, respectively. The MRT was 14.39 ± 0.30 hr. The Vd and CL in brain were 35070.00 ± 2110.32 ml.m-2 and 2258.90 ± 127.06 ml.hr-1.m-2.
The mean brain concentration-time profile of PCB 77 in mouse brain after inhalation of PCB mixture at three different dose levels are presented in Figure 1, inset B, and Table 1.
Pharmacokinetics profile of PCB 180 at 2.0, 4.0 and 16.0 mg.m-3 in mice plasma:
The mean Cmax of PCB 180 in plasma after 2 mg.m-3 inhalation of Aroclor 1232 was 66.49 ± 5.22 ng.ml-1. The time taken to reach this value of Cmax in plasma was 2.00 hr. The AUC0-t and AUC0-∞ attained by PCB 180 in plasma in this dose level were 763.27 ± 38.80 and 1226.52 ± 136.72 ng.hr.ml-1, respectively. The Elimination rate constant of PCB 180 for its elimination from plasma at this dose level was 0.02 hr-1. Its half-life in plasma was 28.95 ± 3.39 hr. Its MRT in plasma was 13.11 ± 0.96 hr at this dose level. The plasma Vd and CL of PCB 180 after administration of 2 mg.m-3 of Aroclor 1232 for 6 hr were 68083.41 ± 1151.95 ml.m-2, and 1646.03 ± 184.36 ml.hr-1.m-2.
The mean Cmax of PCB 180 in plasma in the 4 mg.m-3 group was 370.21 ± 42.59 ng.ml-1, and the time taken to reach this concentration in plasma (Tmax) was 2.00 hr. The AUC0-t and AUC0-∞ were 4369.75 ± 730.55 and 5425.39 ± 408.95 ng.hr.ml-1. This congener’s elimination rate constant and half-life were 0.04 ± 0.01 hr-1 and 20.04 ± 6.43 hr, respectively. MRT in plasma for this group was 15.43 ± 0.85 hr; with a Vd and plasma CL of 21729.82 ± 8734.97 ml.m-2 and 740.51 ± 57.37 ml.hr-1.m-2, both somewhat less compared to the lower dose level.
In the 16 mg.m-3 exposure, the time taken to reach a plasma Cmax of 829.57 ± 31.40 ng.ml-1 was 6.00 hr. The corresponding AUC0-t and AUC0-∞ for the plasma-concentration-time relationship in this group were 13192.39 ± 498.68 and 15636.03 ± 1053.10 ng.hr.ml-1. The Kel and t-1/2 in this group were 0.04 ± 0.01 hr-1 and 18.21 ± 1.94 hr, respectively, and the MRT was 16.24 ± 0.32 hr. The Vd and CL of PCB 180 from plasma at this dosing level were 26825.47 ± 1287.33 ml.m-2 and 1026.94 ± 72.53 ml.hr-1.m-2, respectively.
The mean plasma concentration-time profile of PCB 180 in mouse plasma after inhalation of PCB mixture at three different dose levels are presented in Figure 2, inset A, and Table 2.
Pharmacokinetics profile of PCB 180 at 2.0, 4.0 and 16.0 mg/m3 in mice brain:
The peak concentration of PCB 180 attained in the brain in the 2, 4 and 16 mg.m-3 groups were42.04 ± 4.83, 135.49 ± 4.95 and 429.35 ± 14.37 ng.ml-1, respectively, showing a geometrically progressive trend with dose. The Tmax of this congener in brain were 2.0 ± 0.0, 2.0 ± 0.0, and 6.0 ± 0.0 hr in the three dose levels, respectively. The AUC0-t in brain were 631.12 ± 46.60, 2449.37 ± 186.79, and 8332.99 123.54, ng.hr.ml-1 respectively with dose increments; the AUC0-∞ for PCB 180 across the three dose levels were 915.48 ± 106.63, 3896.91 ± 367.99, and 10984.01 ± 590.37 ng.hr.ml-1, respectively. The Kel in brain were 0.03, 0.02 and 0.03 hr-1 across the three graded doses of Aroclor 1232. The half-lives of PCB 180 elimination from brain were 22.17 ± 3.24, 31.43 ± 3.10, and 22.73 ± 3.57 hr, while the MRT were 13.88 ± 0.21, 19.45 ± 0.64, and 18.72 ± 0.21 hr correspondingly, in the brain. The mean apparent Vd of PCB 180 in the mouse brains in the 2, 4 and 16 mg.m-3 doses were 69838.36 ± 5306.26, 46934.13 ± 6867.21 and, 47593.68 ± 4968.74 ml.m-2 and its clearance were 2207.04 ± 256.46, 1033.24 ± 96.14 and 1459.71 ± 75.49 ml.hr-1.m-2.
The mean brain concentration-time profile of PCB 180 in mouse brain after inhalation of PCB mixture at three different dose levels are presented in Figure 2, inset B, and Table 2 (Supplemental files 1A and 1B).
Dose dependence of plasma PCB 180 and PCB 77:
Aroclor 1232 was dosed to Swiss albino mice at three dose levels – 2.0 (low dose), 4.0 (intermediate dose) and 16.0 (high dose) mg.m-3. PCB 77 Cmax in plasma was eight times higher in high dose compared to low dose level. However, this was only three times higher in intermediate dose group compared to 2.0 mg/m3 dose level and three times higher for high dose compared to 4.0 mg/m3. In plasma, PCB 180 Cmax was six times and twelve times higher for intermediate and high dose groups compared to 2.0 mg/m3 dose levels. The high dose level Cmax for plasma PCB 180 was twice than intermediate dose level.
Dose-dependent translation of brain to plasma PCB 77, 180 ratios:
PCB 77 brain to plasma ratios were similar for intermediate and high dose level, which reached about 1.1 times for lower dose level. The ratios for PCB 180 were three times in the intermediate dose level compared to high dose; the ratio was 5 times higher in low dose compared to the high dose level, and 1.7 times high in intermediate dose compared to low dose (Figure 3, Table 3, Supplemental files 2A and 2B).
Effect of brain to plasma ratio of PCB 77 and PCB 180 in Swiss albino mice:
Hill model was used to quantify the Inhibitory Dose, ID-50 values for PCB 180 and PCB 77 to depress behavioral scores at the same level of brain to plasma ratios (Figure 3, and, 4). PCB 180 ID-50 was 7.645 mg.m-3, and this was higher than PCB 77 ID-50 values (7.382 mg.m-3). The slopes of lines were 13.89 and 12.42, and Y-Intercepts for behavioral disruption were 38.23 and 37.16 respectively for PCB 77 and PCB 180.
Effects of Aroclor 1232 on behavior:
There was a significant difference in total scores at 3 and 4 hours for all three doses. Significant difference in mean scores was also observed at 5 hours of single-6-hour exposures for 2 and 4 mg.m-3 doses (p< 0.05), when compared to control group. The sub scores that contributed to greater than 50% deviation from the normative values, as assessed by Mann Whitney U test were distributed across three main domains – mood, autonomic activity, and miscellaneous functions, for all dose levels. The Cmax of 2, 4, and 16 mg.m-3 dose groups were achieved at 3.5 ± 1.00, 4.0 ± 0 and 4.0 ± 0 hr for PCB 77 in plasma and brain. The Tmax of PCB 180 plasma and brain at 2, 4, and 16 mg.m-3 dose levels were 2.0 ± 0, 2.0 ± 0 and 6.0 ± 0 hr. A more detailed analysis of subdomain score across doses revealed that grooming and restlessness (mood), urination, heart and respiratory rates, skin colour and palpebral opening (autonomic), and exploratory and group clustering behaviors (miscellaneous domain) were contributing to the statistical significance in the behavioral changes compared to control group (Supplemental files 3A and 3B).
Brain monoamine concentrations at Tmaxof PCB congeners after exposures to different doses of Aroclor 1232:
The brain concentrations of Noradrenaline at 4 hr (Cmax of PCB 77 in brain) were 3.8, 1.7 and 2.0 ng/ml across 2, 4, and 16 mg.m-3doses. Dopamine (DA) reached 4.8, 1.6 and 7.5 ng/ml in 2, 4 and 16 mg.m-3doses at 4 hr. (Tmax of PCBs in brain).At 4 hr corresponding to Tmax of PCBs in brain, the brain 5-HT levels were 3.0, 2.6 and 4.3 ng.ml-1 in 2, 4 and 16 mg.m-3doses. Brain levels of Noradrenaline and Dopamine were reduced as PCB 77 concentrations reached maximum in brain. PCB 180 levels did not correlate with either central behavioral disruption or brain levels of all three monoamines.
Effects of Aroclor 1232 on brain monoamine levels: Relationship with behavioral scores:
Brain monoamine levels were estimated using HPLC-FLD in the first six hours for each dose level. Brain monoamine levels followed a linear relationship for 5-HT, exponential relationship for NA and non-monotonic or bimodular for DA in the first six hours of exposures. These could be correlated with behavioral scores at Cmax time points for each dose level (Figure 5, Table 4, Table 5).
Generally, a decline in mood was noted due to dipping of scores for grooming and restlessness sub-domains. The mean grooming scores across all days dipped at 5 hours for higher dose levels as compared to 4 and 6 hours for 2 mg.m-3 dose. The trough in mean restlessness scores across all days was 3 hours for low dose and this shifted by one hour for each geometric increase in dose (Supplemental files 4A, 4B and 4C).
In 2 mg.m-3exposed animals, the mood was generally depressed due to reduction in grooming at 3 and 4 hours, and increase in restlessness at 3, 4, and 5-hour time points. Grooming scores was back to control values at 5 hours in this dose, whereas increased restlessness did not recover even at 6 hours in this group. In the 4 mg/cu.m dose groups, grooming scores dipped at 3, 4, and 5-hour time points. But restlessness scores increased at 3, 4 and 5-hour time points. In the 16 mg.m-3dose level, the grooming reduction was maximum at 3, 4, and 5 hr after exposure and did not recover even at 6 hr in this dose level. Restlessness was increased 3 hr, after which it came to normal at 4 hr time point in this dose level. At 5 hr after exposure, the restlessness scores increased, which did not recover back in this dose level.
In this study, the mean palpebral opening was below the normative value at all time points, with the maximum dip approached at 4 hours for 2 mg.m-3dose and at 5 hours for higher doses. Although the heart rate was below the normative score during all time points, there was a tendency for maximum bradycardia at 3-5 hours across all dose levels. Similarly, the respiratory rate tended to be below the normative always and to initially dip at about 2-3 hours and then recover to baseline value at 5 hours across all days and dose levels. Urination scores increased at 5 hours for 2 and 4 mg.m-3doses across the day averages, whereas it reduced at 4 and 5 hours for the 16 mg.m-3dose.
Among the miscellaneous domains, group clustering (social behavior) activity increased with duration of exposure as the animals tended to reduce the surface area of their fur to avoid exposure to the potentially irritating PCBs. This was inversely proportional to the exploratory activity of mice in the chamber. Exploratory activity increased at 3, 4, and 5 hr in 2, 4, and 16 mg.m-3dose levels. Social behavior increased at 3, 4, and 5 hr in 2 and 16 mg.m-3dose levels. However, in the 4 mg.m-3dose level, the social behavior scores were normal at 3 hr, then it decreased at 4 hr, after which it again recovered back to normal at 5 hr (Table 5).
Relationship of sub-domain behavior scores across doses:
The total behavioral subdomain scores across each time point compared pair wise during single 6-hour exposure to Aroclor 1232 exhibited dose dependence at 2.0, 4.0 and 16.0 mg.m-3dose levels (F ratio = 18.41, p < 0.0001). The Pearson Correlation coefficients were close to 1.0 for most of the pair-wise comparisons. One-way Repeated Measures ANOVA of each correlation coefficients showed that the significance was due to inter-dose variation than inter-individual variability (p = 0.0077).
Dose dependence of sub-domain scores at Tmax:
Likewise, the behavior scores were statistically significant at Tmax of PCB 77 and PCB 180 (3-5 hours) with p value 0.0011. Friedman test of the correlation coefficients at Tmax was significant across doses (Friedman statistic = 31.04). There was more of behavioral depression than stimulatory effects of PCBs at Tmax.
Contribution of sub-domains to dose-dependent behavioral effects of PCBs at Tmax:
Mood, which composed of five sub-domains namely grooming, restlessness, vocalization, irritability, and fearfulness, was generally depressed across all doses of exposures. This was generally ascribed to depression in grooming behavior on Irwin scale across all three doses, although restlessness seemed to increase dose-dependently. Concentrations of PCB 77 and PCB 180 in lung, brain and plasma significantly correlated negatively with mood depression at 6 hours in each dose level (-0.75 <Pearson ‘r’< -0.85, Figure 6). Grooming depression was maximum at 4 hours and Restlessness was maximally increased at 4 hours across all doses of exposure (Figure 7).
Of the eleven autonomic system functions including palpebral opening, writhing response, pupil size, exophthalmos, pupil size, urination, salivation, hypothermia, skin colour, heart rate and respiratory rate, palpebral opening, urination and skin colour increased at 5 hours of exposure, especially at 4.0 and 16.0 mg.m-3 doses. However, heart rate and respiration, reduced at 3 hours maximally across all three doses.
Out of the three miscellaneous domains (social aggregation, hostility, and exploratory activity), social conglomeration increased at 3-5 hours across all three doses, and exploratory activities increased at 3-5 hours across 2.0 and 16.0 mg.m-3 dosing groups.
Generally, the peak of central behavioral disruption followed the Tmax of PCB 77, whereas PCB 180 Cmax contributed more to the peripheral effects on behavior (Table 4, and, Table 5, Supplemental files 1A, 1B, and 5).