Document DDekExV5M8z2b8235EX2pjd9o
Report Summary
Indigo Biosciences Study Report 110407-1
3M ToxDocs 11-108
Prepared by: John L. Butenhoff, Ph.D., D.A.B.T., C.I.H.
Sue Chang, Ph.D. July, 2011
BACKGROUND:
Various toxicological studies in rodents have shown that perfluoroalkyl acids (PFAs) produce responses that are consistent with activation of the xenosensor nuclear receptors peroxisome proliferator activated receptor (PPAR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) (Bjork and Wallace 2009; Curran et al. 2008; Martin et al. 2007; Pieterman et al. 2007; Ren et al. 2009; Rosen et al. 2010; Rosen et al. 2009; Shipley et al. 2004; Takacs and Abbott 2007; Vanden Heuvel et al. 2006; Wolf et al. 2008). The activations of these xenosensor nuclear receptors can lead to hepatocellular hyperplastic responses in rodents. Even though
human hepatocytes contain active forms of PPAR and CAR/PXR receptors, there are significant differences exist in pharmacokinetic / pharmacodynamic profiles between the human and rodent forms. To date, a number of studies have shown that the hyperplastic component of the response to inducers of xenosensor nuclear receptors observed in rodents appears unlikely to be a component of the human hepatic response (Ross et al. 2010). In the evaluation of PFAs of
various chain lengths, Wolf et al. (2008) demonstrated that activation of mouse PPAR was
generally higher compared to that of human PPAR using transfected COS-1 cells. Bjork and Wallace (2009) also reported that primary rat hepatocytes were more effective in eliciting
PPARrelated responses than primary human hepatocytes. Furthermore, with the 8-carbon perfluoroalkyls perfluorooctanesulfonate(PFOS) and perfluorooctanoate (PFOA), Bjork et al.
(2011) showed that while multiple xenosensor nuclear receptors (i.e., PPAR, CAR, PXR) participate in the metabolic response to PFOA and PFOS exposures in both human and rat hepatocytes, however, the changes in primary human hepatocytes were more subtle and possibly reflect an adaptive metabolic response rather than an overt metabolic regulation observed in rodents.
OBJECTIVE:
The objective of the work reported herein was to investigate the effects of perfluorobutyrate (PFBA), PFOA, perfluorobutanesulfonate (PFBS), pherfluorohexanesulfonate (PFHxS), and PFOS on the activation of three human xenosensor nuclear receptors (thyroid receptors alpha (TR), PXR, and CAR3) using HEK-293T transactivated cells.
MATERIALS AND METHODS:
The test was performed by Indigo Biosciences. The compounds and concentrations evaluated are:
Compound PFBA, potassium salt PFOA, ammonium salt PFBS, potassium salt PFHxS, potassium salt PFOS, potassium salt PFOS, potassium salt
Lot # NB # 119868-94 (FC-143) Lot # 332 (L-7038) Lot # 2 (L-9051) NB # 118993-109 (FC-95) Lot # 217 (FC-95) TCR 00017-46
Concentrations Tested 0.41 - 100 M 0.41 - 100 M 0.41 - 100 M 0.41 - 100 M 0.41 - 100 M 0.41 - 100 M
RESULTS:
The full report from Indigo Biosciences is attached as Appendix I of this report. The table below summarizes the study results.
Xenosensor Nuclear Receptor
Compound
Dose Range (M)
Highest Peak Response
EC50 (M)
p-value
T3a (+ control)
0 - 0.01
80.43
0.002123
--
PFOS
0.41 - 100
1.36
0.9528
0.0087
PFOS (lot 217)
0.41 - 100
1.475
7.466
0.0014
TR
PFHxS
0.41 - 100
1.623
17.46
0.0015
PFBS
0.41 - 100
1.224
2.534
0.0035
PFOA
0.41 - 100
1.542
NAb
0.1817
PFBA
0.41 - 100
0.978
1.758
0.3298
TO-9013173c (+ control)
0 - 1
8.343
0.03377
--
PFOS
0.41 - 100
1.706
8.172
0.0032
PFOS (lot 217)
0.41 - 100
2.045
122.2
0.0018
PXR
PFHxS
0.41 - 100
2.81
47.25
0.0005
PFBS
0.41 - 100
1.693
18.28
0.0008
PFOA
0.41 - 100
1.07
NA
0.0765
PFBA
0.41 - 100
0.966
NA
0.0314
CITCOd (+ control)
0 - 5
37.042
0.6444
--
PFOS
0.41 - 100
2.025
38.11
0.0001
PFOS (lot 217)
0.41 - 100
2.547
525.9
0.0021
CAR3
PFHxS
0.41 - 100
1.963
62.41
0.0516
PFBS
0.41 - 100
1.13
3.247
0.0525
PFOA
0.41 - 100
1.723
NA
0.0275
PFBA
0.41 - 100
1.15
3.885
0.128
a T3, triiodothyronine, agonist for TR. b NA, not available, EC50 cannot be derived because data did not fit the curve. c TO-9013173, agonist for PXR.
d CITCO, 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehydeO-(3,4-dichlorobenzyl)oxime,
agonist for CAR3.
Human thyriod receptor R:
There were statistical associations suggesting that PFBS, PFHxS, and PFOS were capable
of eliciting human R activation in the assay. However, unlike the positive control triiodothyronine (T3), there were no concentration-dependent responses with any of the PFAs evaluated in the activation of human TR and the EC50's were at least several
orders of magnitude higher than T3. Therefore, PFAs does not have any effect on R activation.
Human pregnane X receptor (PXR):
Within the limit of study design, PFOS (lot 217) and PFHxS at 33 M or higher appeared to elicit some human PXR responses, although the EC50's derived were several orders of magnitude higher than the positive control. PFBS did not have any effect on PXR activation.
Even though the data obtained from PFOA and PFBA did not fit the statistical curves and no reliable EC50 could be derived, given the fact that the highest responses obtained with either PFOA or PFBA were at least an order of magnitude lower than the highest response obtained from positive control, it is unlikely that PFOA or PFBA is capable of activating human PXR.
Human Constitutive androstane receptors 3 (CAR3):
PFOS (both lot 217 and TCR 00017-46) at 100 M (the highest concentration tested) were capable of eliciting human CAR3 responses. Similar to PXR, the EC50's derived were several orders of magnitude higher than the positive control. PFBS and PFBA did not have any effect on human CAR3 activation.
Due to the lack of concentration-dependent response, the data obtained from PFOA did not fit the statistical curves and no reliable EC50 could be derived, given the fact that the highest response obtained were much lower than the highest response obtained from positive control, it is unlikely that PFOA activates human CAR3.
CONCLUSION:
PFAs do not appear to activate human TR. Consistent with other study data reported by others, PFOS appears to be capable of eliciting human PXR and CAR3 responses in vitro. However, the degree of activation was several folds lower than the activation produced by TO-9013173 and CITCO (the respective controls for PXR and CAR3), and the concentrations of PFOS employed in the assay were several folds higher than the blood serum PFOS concentrations reported for general human population. PFHxS also appears to be capable of eliciting human PXR responses as well, and similar to PFOS, the extent of activation was much lower than the respective control and the concentrations of PFHxS that produced effects were much higher than the general human population. PFBS, PFOA, and PFBA did not activate human PXR and CAR3.
REFERENCES:
Bjork, J.A., Wallace, K.B., 2009. Structure-activity relationships and human relevance for perfluoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures. Toxicol Sci 111, 89-99.
Curran, I.,Hierlihy, S.L.,Liston, V.,Pantazopoulos, P.,Nunnikhoven, A.,Tittlemier, S.,Barker, M.,Trick, K., Bondy, G., 2008. Altered Fatty Acid Homeostasis and Related Toxicologic Sequelae in Rats Exposed to Dietary Potassium Perfluorooctanesulfonate (PFOS). J Toxicol Environ Health A 71, 1526-1541.
Martin, M.T.,Brennan, R.J.,Hu, W.,Ayanoglu, E.,Lau, C.,Ren, H.,Wood, C.R.,Corton, J.C.,Kavlock, R.J., Dix, D.J., 2007. Toxicogenomic study of triazole fungicides and perfluoroalkyl acids in rat livers predicts toxicity and categorizes chemicals based on mechanisms of toxicity. Toxicol Sci 97, 595-613.
Pieterman, E.,van den Hoek, A.M.,Havekes, L.M.,Ehresman, D.,Chang, S.,Butenhoff, J.L.,Princen, H.M., Cohen, L.H., 2007. Perfluorohexanesulfonate and perfluorooctanesulfonate decrease plasma cholesterol and triglycerides in APOE*3Leiden transgenic mice. Indication for a PPAR agonist mechanism (abstract). Atherosclerosis Suppl 8, 41.
Ren, H.,Vallanat, B.,Nelson, D.M.,Yeung, L.W.Y.,Guruge, K.S.,Lam, P.K.S.,LehmanMcKeeman, L.D., Corton, J.C., 2009. Evidence for the Involvement of Xenobioticresponsive Nuclear Receptors in Transcriptional Effects Upon Perfluoroalkyl Acid Exposure in Diverse Species. Reproductive Toxicology 27, 266-277.
Rosen, M.B.,Schmid, J.E.,Das, K.P.,Wood, C.R.,Zehr, R.D., Lau, C., 2009. Gene Expression Profiling in the Liver and Lung of Perfluorooctane Sulfonate-Exposed Mouse Fetuses: Comparison to Changes Induced by Exposure to Perfluorooctanoic Acid. Reproductive Toxicology 27, 278-288.
Rosen, M.B.,Schmid, J.E.,Corton, J.C.,Zehr, R.D.,Das, K.P., Lau, C., 2010. Gene Expression Profiling in Wild-Type and PPAR-Null Mice Exposed to Perfluorooctane Sulfonate Reveals PPAR-Independent Effects. PPAR Research 2010, 1-23.
Ross, J.,Plummer, S.M.,Rode, A.,Scheer, N.,Bower, C.C.,Vogel, O.,Henderson, C.J.,Wolf, C.R., Elcombe, C.R., 2010. Human constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) Support the Hypertrophic but not the Hyperplastic Response to the Murine Nongentoxic Hepatocarcinogens Phenobarbital and Chlordane In Vivo. Toxicol Sci 116, 452466.
Shipley, J.M.,Hurst, C.H.,Tanaka, S.S.,DeRoos, F.L.,Butenhoff, J.L.,Seacat, A.M., Waxman, D.J., 2004. trans-activation of PPARalpha and induction of PPARalpha target genes by perfluorooctane-based chemicals. Toxicol Sci 80, 151-160.
Takacs, M.L., Abbott, B.D., 2007. Activation of mouse and human peroxisome proliferatoractivated receptors (alpha, beta/delta, gamma) by perfluorooctanoic acid and perfluorooctane sulfonate. Toxicol Sci 95, 108-117.
Vanden Heuvel, J.P.,Thompson, J.T.,Frame, S.R., Gillies, P.J., 2006. Differential Activation of Nuclear Receptors by Perfluorinated Fatty Acid Analogs and Natural Fatty Acids: A Comparison of Human, Mouse, and Rat Peroxisome Proliferator-Activated Receptor-and -Liver X Receptor-, and Retinoid X Receptor-. Toxicological Sciences 92, 476-489.
Wolf, C.J.,Takacs, M.L.,Schmid, J.E.,Lau, C., Abbott, B.D., 2008. Activation of mouse and human peroxisome proliferator-activated receptor alpha by perfluoroalkyl acids of different functional groups and chain lengths. Toxicol Sci 106, 162-171.
APPENDIX I: Study Report from Indigo Biosciences
Indigo Biosciences, Inc Report 110407-1 3M Summary April 7, 2011
SUMMARY
Effects of test compounds on nuclear receptor activation:
TRalpha: PXR: CAR3:
No effects PFOS217 at 100 M; PFHxS at 33 and 100 M PFOS at 100 M; PFOS217 at 100 M
EXPERIMENTAL PROCEDURE
Plasmids:
Two different assays are commonly used for services, a chimeric assay and a full- length (see Figure 1).
Chimeric System: The ligand-binding domain of the nuclear receptors were fused to the DNA-binding domain of the yeast transcription factor Gal4
under the control of the SV40 promoter. A reporter plasmid encodes the firefly luciferase gene under the control of the Gal4 DNA response element (UAS). A transfection efficiency control vector is included in most assays (pRL-luciferase, Promega, Madison, WI). All plasmids were verified by sequencing and through examination of positive controls (Tien et al., 2006; Vanden Heuvel et al., 2006).
Full Length System: The full-length cDNA of the nuclear receptor is under the control of the SV40 promoter. A reporter plasmid encodes the luciferase
reporter under the control of the MMTV response element. All plasmids were verified by sequencing and through examination of positive controls.
Figure 1. Design of Reporter Assays Performed by INDIGO Biosciences, Inc. Cell culture and transactivation assays:
HEK 293-T fibroblasts (ATCC, Manassas, VA) were cultured in high-glucose Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma), 0.2 mg/ml streptomycin and 200 U/ml penicillin (Gibco, Grand Island, NY). For transient transfection reporter assays, HEK 293-T ells were transfected with plasmid DNA using Lipofectamine reagent (Invitrogen, Carlsbad, CA) and following the manufacturer's recommended procedures, using HEK 293-T cells at approximately 80% confluence in 10 cm culture dishes. After 6 h, the DNA- Lipofectamine complex was removed. Following overnight culture, the media was replaced 4 h after replating with DMEM (10% FBS) containing test compounds in DMSO (0.1% final concentration). Concentrations of the chemicals are given in the figure legends. Sixteen hours after treatment, the cells were lysed with passive lysis buffer (Promega, Madison, WI) for 30 min; luciferase activity was measured using the Luciferase dual reporter assay kit (Promega, Madison, WI) and a Tecan GeniosPro (Research Triangle Park, NC) and manufacturer's recommended procedures. The fold induction of normalized luciferase activity (luciferase/ renilla) was calculated relative to vehicle-treated cells, and represents the mean of three independent samples per treatment group.
Statistical Analysis:
Significance of dose-dependent alterations in relative luciferase activity (p<0.05) was determined using ANOVA (p<0.05, JMP 7.0, SAS Institute, Cary,NC). Dunnett's multi-comparison t-test was used to determine doses that resulted in significant differences relative to vehicle control treated cells (JMP 7.0). Non-linear regression was performed using Prism 5.0 (Graphpad Software, La Jola, CA).
References:
Tien, E.S., Hannon, D.B., Thompson, J.T., and Vanden Heuvel, J.P. (2006). Examination of Ligand-Dependent Coactivator Recruitment by Peroxisome Proliferator-Activated Receptoralpha (PPARalpha). PPAR Res 2006, 69612.
Vanden Heuvel, J.P., Thompson, J.T., Frame, S.R., and Gillies, P.J. (2006). Differential activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty acids: a comparison of human, mouse, and rat peroxisome proliferator-activated receptor-alpha, -beta, and -gamma, liver X receptor-beta, and retinoid X receptor-alpha. Toxicol Sci 92, 476-489.
Results:
(A) Human TRalpha Activity Assay
RAW DATA
Human TRalpha Activity Assay SUMMARIZED DATA
Group
Compound 1 DMSO 2 T3 3 T3 4 T3 5 T3 6 T3 7 T3 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS
Dose1 0 3.2 16 80 400 2000 10000 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23
Luc12 0.7829 1.3911 1.6808 2.8709
13.4186 49.8262 97.6308
1.5336 1.6208 1.7300 1.4320 2.0059 1.3598 1.4301 1.4540 1.3263 1.4341 1.1855 2.0159 1.7592 1.0655 1.3552 1.2197
Luc2 1.2260 1.4683 1.7300 2.4996
13.5485 57.6902 88.6182
1.1860 1.3368 1.6780 1.8906 1.7053 1.5801 1.4621 1.4147 1.3898 1.6616 1.6524 2.1002 1.8390 1.2300 1.1638 1.1961
Luc3 1.0826 1.2120 1.6432 2.3047
14.5054 62.8881 87.9161
1.2438 1.2753 1.6351 1.7369 1.8423 1.3423 1.1920 1.3160 1.5206 1.6981 1.6048 1.9061 1.5170 0.9756 1.2255 0.9992
Group
Compound 1 DMSO 2 T3 3 T3 4 T3 5 T3 6 T3 7 T3 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS
Dose
Mean3
Std Err
CV
0
1.0000
0.0398
3.2
1.1945
0.0668
16
1.4827
0.0221
80
2.2518
0.1462
400 12.1671
0.3016
2000 49.9929
3.3416
10000 80.4340
2.7529
0.41
0.9704
0.0790
1.23
1.0364
0.0782
3.7
1.2348
0.0202
11.1
1.2388
0.0990
33.3
1.3598
0.0638
100
1.0485
0.0562
0
1.0000
0.0626
0.41
1.0246
0.0302
1.23
1.0373
0.0420
3.7
1.1738
0.0607
11.1
1.0878
0.1090
33.3
1.4745
0.0413
100
1.2524
0.0711
0
1.0000
0.0683
0.41
1.1447
0.0517
1.23
1.0440
0.0641
6.8851 9.6853 2.5819 11.2433 4.2934 11.5774 5.9280 14.0966 13.0617 2.8275 13.8422 8.1285 9.2833 10.8415 5.0976 7.0181 8.9535 17.3490 4.8480 9.8333 11.8314 7.8284 10.6380
Stats4 0.0087 0.0014 0.0015
p-value5
0.9993 0.9978 0.1273 0.1186 0.012 0.9904
1 0.9996 0.9963 0.2795
0.837 0.0007 0.0641
1 0.7995 0.9991
Renilla6
0.90724986 1.00495683 0.92482502 0.78996843 0.5186502 0.61092297
1 1.00549124 1.06633562 0.94738703 0.82781468 0.65097695 0.63612893
1 0.98896434 0.99012433
24 PFHxS
3.7 1.02835 1.223711 1.431821
24 PFHxS
3.7 1.12618328 0.10683624 16.4312322
0.8723 0.99138284
25 PFHxS
11.1 1.392872 1.459168 1.302646
25 PFHxS
11.1 1.27011069 0.04160013 5.67301272
0.2744 1.02595598
26 PFHxS
33.3 1.623792 1.799535 1.884285
26 PFHxS
33.3 1.6225666 0.07035373 7.51009139
0.0028 0.79998882
27 PFHxS
100 1.75969 1.39303 2.142978
27 PFHxS
100 1.61892442 0.1985641 21.243926
0.0029 0.54180085
28 PFBS
0 1.035781 1.261699 1.234255
28 PFBS
0
1 0.06045835 10.4716941
0.0035
1
1
29 PFBS
0.41 1.004704 1.079429 1.217323
29 PFBS
0.41 0.9347972 0.05289827 9.80132274
0.9307 0.8384736
30 PFBS
1.23 0.7317497 1.077492 1.036778
30 PFBS
1.23 0.8058418 0.0926712 19.9184531
0.1556 0.66056222
31 PFBS
3.7 1.280438 1.577579 1.464072
31 PFBS
3.7 1.22378633 0.07353596 10.4077002
0.0849 0.83033015
32 PFBS
11.1 1.188956 1.45895 1.279778
32 PFBS
11.1 1.11211175 0.06737708 10.4935965
0.6166 0.92584538
33 PFBS
33.3 1.347046 1.306689 1.344105
33 PFBS
33.3 1.13197621 0.01103419 1.68835449
0.4681 0.85057157
34 PFBS
100 1.301196 1.379461 1.349161
34 PFBS
100 1.14103068 0.01935382 2.93785192
0.4068 0.69897459
35 PFOA
0 1.208199 1.082189 1.120016
35 PFOA
0
1 0.03283914 5.68790617
0.1817
1
1
36 PFOA
0.41 1.162493 1.788925 1.337093
36 PFOA
0.41 1.25747888 0.16418436 22.6147452
0.5882 1.11237482
37 PFOA
1.23 1.45962 1.604089 1.301355
37 PFOA
1.23 1.27992578 0.07690176 10.4066779
0.5121 1.24511386
38 PFOA
3.7 1.917233 1.412162 1.929093
38 PFOA
3.7 1.54189592 0.14986618 16.8348481
0.0555 1.1221669
39 PFOA
11.1 1.41738 1.93721 1.387912
39 PFOA
11.1 1.3905983 0.15692358 19.5455164
0.2224 1.15636803
40 PFOA
33.3 1.250512 1.199197 1.943313
40 PFOA
33.3 1.28812364 0.21106931 28.3810304
0.4853 0.98125502
41 PFOA
100 1.214905 1.29889 1.253875
41 PFOA
100 1.10475768 0.02134525 3.34653057
0.9835 0.67508295
42 PFBA
0 1.498251 1.936381 1.693365
42 PFBA
0
1 0.07413945 12.84133
0.3298
1
1
43 PFBA
0.41 1.694401 1.747789 1.292758
43 PFBA
0.41 0.92335233 0.08401431 15.7596455
0.8183 1.01709165
44 PFBA
1.23 1.639416 1.554529 1.692198
44 PFBA
1.23 0.95283656 0.02345958 4.26444437
0.9727 1.14804843
45 PFBA
3.7 1.778415 1.648278 1.589801
45 PFBA
3.7 0.97825603 0.03261079 5.77390251
0.9995 1.02335903
46 PFBA
11.1 1.413671 1.450736 1.361416
46 PFBA
11.1 0.82406893 0.01515706 3.18575257
0.1511 1.00881868
47 PFBA
33.3 1.434516 1.443324 1.613287
47 PFBA
33.3 0.87580531 0.03403547 6.73108011
0.4261 1.12207964
48 PFBA
100 1.662825 1.405156 1.822196
48 PFBA
100 0.95362322 0.07107962 12.9100795
0.9747 1.094378
1 Dose in pM for T3 and uM for client's compounds
3 Expressed relative to DMSO control
6 Relative Renilla activity (toxicity marker)
2 Expressed as (Luciferase RLU/Renilla RLU)
4 Oneway Anova, Dose x Relative Luciferase, Prob > F
Relative renilla below 50% (0.5) indicates overt toxicit
5 Dunnett's t-test, DMSO control versus each sample
Curve Fitting Data
T3
PFOS PFOS217 PFHxS
PFBS
PFOA
PFBA
Bottom
-0.07873
0.9065
0.9974
1.028
0.9063 Ambiguous
0.9826
Top
98.35
1.233
1.366
1.779
1.167
0.894
LogEC50
3.327 -0.02099
0.8731
1.242
0.4038
0.245
EC50
2123
0.9528
7.466
17.46
2.534
1.758
Span
98.43
0.327
0.369
0.7513
0.261
-0.08855
Statistical Analysis
P-value Peak4 LSSD5 4 Highest observable 5 Lowest statistically NA, not applicable
PFOS
PFOS217 PFHxS
0.0087
0.0014
0.0015
1.360
1.475 1.6225666
33.3
33.3
33.3
response (relative to DMSO control)
significant dose (p<0.05, Dunnett's)
PFBS
0.0035 1.2237863 N/A
PFOA
0.1817 1.5418959 N/A
PFBA
0.3298 0.8240689 N/A
Relative Luciferase Activity Relative Luciferase Activity
100
T3 80
60
40
20
0
0
1
2
3
4
5
Log(Dose) pM
3 PFOS
PFOS217
2
PFHxS
PFBS
PFOA
1
PFBA
0
-1
0
1
2
Log(Dose) M
(B) Human PXR Activity Assay
RAW DATA
Group
Compound 1 DMSO 2 TO-9013173 3 TO-9013173 4 TO-9013173 5 TO-9013173 6 TO-9013173 7 TO-9013173 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS 24 PFHxS 25 PFHxS 26 PFHxS 27 PFHxS 28 PFBS 29 PFBS 30 PFBS 31 PFBS 32 PFBS 33 PFBS 34 PFBS 35 PFOA 36 PFOA 37 PFOA 38 PFOA 39 PFOA 40 PFOA 41 PFOA 42 PFBA 43 PFBA 44 PFBA 45 PFBA 46 PFBA 47 PFBA 48 PFBA
Dose1 0 0.32 1.6 8 40 200 1000 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100
Luc12 0.2699435 1.423668 2.186356 2.075191 4.315969 7.138336 6.916809 1.451643 1.790085 1.87229 1.613219 2.22789 2.704702 1.913311 1.616936 1.555113 2.453635 1.455274 2.625709 3.184645 1.519225 1.513617 1.227581 0.9261827 2.194648 2.512845 2.747372 1.234832 2.301728 2.631239 1.931713 1.639419 1.359096 1.097309 2.475862 0.8823314 2.016315 1.732709 1.730784 1.061295 3.558488 1.737606 1.564718 1.315794 1.205181 1.082178 1.451322 1.919343
Luc2 0.7931641 2.339388 1.62824 2.24288 4.973182 6.818035 6.96418 1.376961 1.547474 2.037152 3.017765 2.980243 3.347376 1.918895 1.877738 2.457464 2.57857 2.340294 3.331317 3.218487 1.242754 1.4925 1.156105 1.522918 2.146304 2.365294 2.917912 1.597554 2.410897 1.983705 1.97877 1.663126 1.397776 1.191628 2.712058 1.735937 1.389305 2.11884 1.94652 1.556958 2.108 1.728782 1.921795 1.614791 1.655908 1.196779 1.487678 1.283868
1 Dose in nM for TO901317 and uM for client's compounds
2 Expressed as (Luciferase RLU/Renilla RLU)
Human PXR Activity Assay
SUMMARIZED
Luc3 0.9932408 1.035379 1.885377 2.516727 4.966083 7.072407 8.114037 1.484295 1.778647 1.930471 2.610429 2.454123 2.587978 1.231107 1.365523 2.152485 2.489945 2.048763 1.913984 3.950592 0.9651626 1.631546 1.445242 1.737681 1.748068 2.881724 4.808423 1.371582 2.404659 1.791228 1.221857 1.736557 1.278307 1.527382 1.992268 1.606417 0.9668043 1.785514 2.246388 2.084046 2.014824 1.955788 1.171592 1.825245 1.55435 1.458556 1.59692 2.034257
Group
Compound 1 DMSO 2 TO-9013173 3 TO-9013173 4 TO-9013173 5 TO-9013173 6 TO-9013173 7 TO-9013173 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS 24 PFHxS 25 PFHxS 26 PFHxS 27 PFHxS 28 PFBS 29 PFBS 30 PFBS 31 PFBS 32 PFBS 33 PFBS 34 PFBS 35 PFOA 36 PFOA 37 PFOA 38 PFOA 39 PFOA 40 PFOA 41 PFOA 42 PFBA 43 PFBA 44 PFBA 45 PFBA 46 PFBA 47 PFBA 48 PFBA
Dose
0 0.32
1.6 8
40 200 1000 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23
3.7 11.1 33.3 100
0 0.41 1.23
3.7 11.1 33.3 100
0 0.41 1.23
3.7 11.1 33.3 100
0 0.41 1.23
3.7 11.1 33.3 100
0 0.41 1.23
3.7 11.1 33.3 100
Mean3 1
1.82006755 2.16202489 2.59246901 5.40707309 7.97630819 8.34281032 0.85179388 1.01044632 1.15337784 1.43017289 1.51328903 1.70640369
1 0.95988476 1.21759449 1.48561821 1.15425039 1.55451776 2.04485166
1 1.24429482 1.02730951 1.12332241 1.63369699 2.08198771 2.81011781
1 1.69299195 1.52383938 1.22083232 1.19865375 0.95985008 0.90778974
1 0.58838089 0.6089568 0.78508571 0.82500514 0.65489915 1.0697926
1 0.85908407 0.87710727 0.81432971 0.68930131 0.83654975 0.96593471
Std Err 0.06773475 0.43987464 0.18352244 0.14642964 0.24794841 0.11111887 0.44540175 0.01882165 0.04682685 0.02860172 0.24719367 0.13204547 0.13988599
0.1352895 0.08761379
0.1570151 0.0219844 0.1542821 0.24242037 0.1480452 0.12874006 0.03482091 0.06998658 0.19539558 0.11388829 0.12361465 0.53158522 0.07547117 0.02525888 0.18131024 0.17471967 0.02086622 0.02511526 0.09313316 0.08850901 0.11096929 0.12738341 0.05050336 0.062464 0.12337667 0.2088036 0.04107671 0.1198679 0.08177771 0.07552401 0.06162773 0.02420658 0.12910719
3 Expressed relative to DMSO control
4 Oneway Anova, Dose x Relative Luciferase, Prob > F
5 Dunnett's t-test, DMSO control versus each sample
DATA
CV
Stats4
p-value5 Renilla6
11.7320024
41.8602721
14.7024301
9.78308966
7.94254565
2.41293994
9.24698541
3.82722316
0.0032
0.9165 0.907249863
8.02679824
1 1.004956829
4.29517767
0.9048 0.924825023
29.9370795
0.1429 0.789968427
15.113403
0.0644 0.518650202
14.1988466
0.009 0.610922973
23.4328283
0.0018
1
1
15.8093494
1 1.005491244
22.3356905
0.7988 1.066335615
2.56311428
0.1484 0.947387028
23.1513406
0.9419 0.827814675
27.0105882
0.0838 0.650976949
12.539873
0.0011 0.636128925
22.2984328
0.0005
1
1
4.84704883
0.9358
0.98896434
11.799785
1 0.990124327
30.1280445
0.9976 0.991382842
12.0744727
0.2659 1.025955982
10.283771
0.0233 0.799988824
32.7649113
0.0004 0.541800852
13.0719904
0.0008
1
1
2.58416215
0.0021 0.838473597
20.6083762
0.0173 0.660562222
24.7882807
0.5181 0.830330148
3.01516253
0.6136 0.925845375
4.53205125
0.9997 0.850571568
17.7696828
0.9732
0.69897459
15.3302107
0.0765
1
1
32.666671
0.1224 1.112374818
36.2315586
0.1506 1.245113855
11.1420164
0.6516 1.122166898
13.1139568
0.8018 1.156368033
32.6301639
0.2345 0.981255019
33.8064073
0.9963 0.675082953
7.11469436 0.031409
1
1
24.1672842
0.702 1.017091648
16.1488967
0.798 1.148048428
16.0636932
0.4594 1.023359028
15.4855875
0.0864 1.008818678
5.01189864
0.5767 1.122079641
23.1506552
0.9995 1.094378002
6 Relative Renilla activity (toxicity marker)
Relative renilla below 50% (0.5) indicates overt toxicity
Curve Fitting Data
TO901317
Bottom
1.449
Top
8.773
LogEC50
1.529
EC50
33.77
Span
7.325
PFOS 0.8992 1.741 0.9124 8.172 0.8413
PFOS217 1.111 3.179 2.087 122.2 2.068
PFHxS 1.044 3.636 1.674 47.25 2.591
PFBS
PFOA
1.401 Interrupted
0.7898
1.262
18.28
-0.6114
PFBA Interrupted
Statistical Analysis
P-value Peak4 LSSD5 4 Highest observable 5 Lowest statistically NA, not applicable
PFOS
PFOS217 PFHxS
0.0032
0.0018
0.0005
1.706
2.045 2.8101178
100
100
33.3
response (relative to DMSO control)
significant dose (p<0.05, Dunnett's)
PFBS
0.0008
1.692992 0.41
PFOA
0.0765
1.0697926 N/A
PFBA
0.031409
0.6893013 N/A
Relative Luciferase Activity Relative Luciferase Activity
10 TO901317
8
6
4
2
0
-1
0
1
2
3
4
Log(Dose) pM
4 PFOS
PFOS217
3
PFHxS
PFBS
2
PFOA
PFBA 1
0
-1
0
1
2
Log(Dose) M
(C) Human CAR3 Activity Assay
RAW DATA
Group
Compound 1 DMSO 2 CITCO 3 CITCO 4 CITCO 5 CITCO 6 CITCO 7 CITCO 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS 24 PFHxS 25 PFHxS 26 PFHxS 27 PFHxS 28 PFBS 29 PFBS 30 PFBS 31 PFBS 32 PFBS 33 PFBS 34 PFBS 35 PFOA 36 PFOA 37 PFOA 38 PFOA 39 PFOA 40 PFOA 41 PFOA 42 PFBA 43 PFBA 44 PFBA 45 PFBA 46 PFBA 47 PFBA 48 PFBA
Dose1 0 1.6 8 40 200 1000 5000 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100 0 0.41 1.23 3.7 11.1 33.3 100
Luc12 0.2167248 1.506333 1.661328 1.616416 6.079296 26.20632 29.71467 1.2954 1.48921 1.943437 2.207644 2.357472 3.579363 1.912326 1.317282 1.540737 1.812527 1.414215 3.215519 3.44258 1.810927 1.250442 1.558483 0.65403 2.704604 1.915768 2.305565 1.079713 1.028022 0.280539 1.663123 1.293376 1.251064 0.8910954 1.089725 1.243024 1.937257 2.095318 2.081964 1.414356 1.591633 1.811601 2.717717 2.286602 1.951135 1.444449 1.750552 2.031571
Luc2 0.6569583 2.135472 1.75017 1.645852 7.039314 22.4475 30.46399 1.461348 1.626203 2.614328 2.396115 2.187248 3.781329 1.875983 2.546865 1.696937 2.455303 2.01943 2.594508 3.481387 1.347109 1.364983 1.117709 1.186113 1.69808 2.378524 1.811519 1.296352 1.083755 0.5447473 1.508355 1.477396 1.159395 1.369629 1.238347 1.567674 1.951602 1.740867 1.814638 1.651225 1.223631 2.122233 1.987632 2.237881 2.10414 1.721626 1.918734 2.429702
1 Dose in nM for TO901317 and uM for client's compounds
2 Expressed as (Luciferase RLU/Renilla RLU)
Human CAR Activity Assay
SUMMARIZED
Luc3 0.9400501 1.036676 1.654854 2.143853 6.824971 22.89514 31.20507 1.263326 1.499532 2.015931 2.389978 2.338161 2.666823 1.163002 1.013073 1.672663 1.985599 1.870738 2.204319 5.685531 1.06981 1.598152 1.35386 1.338007 1.317618 1.878478 4.181846 1.287055 0.9279157 1.172045 0.978928 1.23914 1.332643 1.274268 1.168205 1.695682 0.9804638 2.189428 1.857409 1.860067 1.308074 2.217719 1.445104 2.522597 2.45519 1.657182 1.753327 2.009645
Group
Compound 1 DMSO 2 CITCO 3 CITCO 4 CITCO 5 CITCO 6 CITCO 7 CITCO 8 PFOS 9 PFOS 10 PFOS 11 PFOS 12 PFOS 13 PFOS 14 PFOS217 15 PFOS217 16 PFOS217 17 PFOS217 18 PFOS217 19 PFOS217 20 PFOS217 21 PFHxS 22 PFHxS 23 PFHxS 24 PFHxS 25 PFHxS 26 PFHxS 27 PFHxS 28 PFBS 29 PFBS 30 PFBS 31 PFBS 32 PFBS 33 PFBS 34 PFBS 35 PFOA 36 PFOA 37 PFOA 38 PFOA 39 PFOA 40 PFOA 41 PFOA 42 PFBA 43 PFBA 44 PFBA 45 PFBA 46 PFBA 47 PFBA 48 PFBA
Dose
0 1.6
8 40 200 1000 5000 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23 3.7 11.1 33.3 100
0 0.41 1.23 3.7 11.1 33.3 100
Mean3 1
1.89641876 2.05364197 2.19136708 8.08411556 29.0023171 37.0423262 0.81192113 0.93206527 1.32766776 1.41250206 1.39011284 2.02522423
1 0.98503609 0.99172462 1.26298449 1.07130879 1.61863111 2.54669884
1 0.996625 0.95321637 0.75171849 1.35300624 1.46002716 1.96292155
1 0.82980975 0.54525413 1.13302485 1.09467121 1.02183439 0.96502228
1 1.28890817 1.39271654 1.72343696 1.64575376 1.40882659 1.17935106
1 0.99982118 1.14557739 1.05834494 0.78407144 0.88150309 1.05191616
Std Err 0.10351685 0.3870763 0.03739328 0.20808699 0.35379451
1.4415003 0.52319677 0.03718058 0.02668682 0.1288026 0.03746052 0.03260492 0.20772368 0.14780522 0.28408237 0.02940407 0.11634902 0.11032706 0.17839661 0.44913387 0.15340346 0.07259294 0.09036442 0.14714271 0.29359727 0.11412028 0.51231352 0.05791329 0.03733765 0.21651598 0.16964702 0.05904785
0.040982 0.11976036 0.03683287 0.11559589 0.27573495 0.11718022 0.07113687 0.11047504 0.09549803 0.05978845 0.17980789 0.04287573 0.07276453 0.04084049 0.02711702 0.06657416
3 Expressed relative to DMSO control
4 Oneway Anova, Dose x Relative Luciferase, Prob > F
5 Dunnett's t-test, DMSO control versus each sample
DATA
CV
Stats4
p-value5 Renilla6
17.9296436
35.3527305
3.15376591
16.4471411
7.58017458
8.60880098
2.44639979
7.93163981
0.0001
0.6929 0.907249863
4.95919338
0.9951 1.004956829
16.8033495
0.2118 0.924825023
4.59351654
0.0839 0.789968427
4.06250254
0.1081 0.518650202
17.7653397
0.0001 0.610922973
25.6006153
0.0021
1
1
49.9519863
1 1.005491244
5.135432
1 1.066335615
15.9560482
0.91 0.947387028
17.8372535
0.9999 0.827814675
19.0897107
0.2754 0.650976949
30.5463162
0.0014 0.636128925
26.5702592
0.0516
1
1
12.6160452
1 0.98896434
16.4197521
1 0.990124327
33.9034695
0.9467 0.991382842
37.5848512
0.8067 1.025955982
13.5382502
0.6075 0.799988824
45.2057319
0.064 0.541800852
10.0308766
0.0525
1
1
7.79343841
0.8134 0.838473597
68.7783281
0.075 0.660562222
25.9338761
0.922 0.830330148
9.34288534
0.9822 0.925845375
6.9466146
1 0.850571568
21.4949473
0.9999 0.69897459
6.37964081
0.0275
1
1
15.5339193
0.5002 1.112374818
34.2917544
0.2323 1.245113855
11.7765893
0.0104 1.122166898
7.48670126
0.0224 1.156368033
13.5821104
0.2032 0.981255019
14.0252924
0.8577 0.675082953
10.3556633
0.128
1
1
31.1492104
1 1.017091648
6.48257753
0.6792 1.148048428
11.9083911
0.9909 1.023359028
9.02185598
0.3261 1.008818678
5.32817916
0.8218 1.122079641
10.9618831
0.995 1.094378002
6 Relative Renilla activity (toxicity marker)
Relative renilla below 50% (0.5) indicates overt toxicity
Curve Fitting Data
CITCO
Bottom
0.7363
Top
42.9
LogEC50
2.809
EC50
644.4
Span
42.16
PFOS 0.9705 2.328 1.581 38.11 1.357
PFOS217 1.009 10.65 2.721 525.9 9.645
PFHxS 0.9236 2.601 1.795 62.41 1.677
PFBS
PFOA
0.8209 Ambiguous
1.053
0.5115
3.247
0.2322
PFBA 1.054
0.9223 0.5894
3.885 -0.1318
Statistical Analysis
P-value Peak4 LSSD5 4 Highest observable 5 Lowest statistically NA, not applicable
PFOS
PFOS217 PFHxS
0.0001
0.0021
0.0516
2.025
2.547 1.9629215
100
100 N/A
response (relative to DMSO control)
significant dose (p<0.05, Dunnett's)
PFBS
0.0525 0.5452541 N/A
PFOA
0.0275 1.723437
3.7
PFBA
0.128 0.7840714 N/A
Relative Luciferase Activity Relative Luciferase Activity
40 CITCO 30
20
10
0
-1
0
1
2
3
4
Log(Dose) pM
4 PFOS
PFOS217
3
PFHxS
PFBS
2
PFOA
PFBA 1
0
-1
0
1
2
Log(Dose) M
Statistical Summary:
TRalpha
Statistical Analysis
PFOS
PFOS217 PFHxS
PFBS
PFOA
PFBA
P-value
0.0087
0.0014
0.0015
0.0035
0.1817
0.3298
Peak1
1.360
1.475 1.6225666 1.2237863 1.5418959 0.8240689
LSSD2
33.3
33.3
33.3 N/A
N/A
N/A
Statistical Analysis
PFOS
PFOS217 PFHxS
PFBS
PFOA
PFBA
PXR P-value Peak1
0.0032 1.706
0.0018
0.0005
2.045 2.8101178
0.0008
0.0765 0.031409
1.692992 1.0697926 0.6893013
LSSD2
100
100
33.3
0.41 N/A
N/A
CAR3
Statistical Analysis
PFOS
PFOS217 PFHxS
PFBS
PFOA
PFBA
P-value
0.0001
0.0021
0.0516
0.0525
0.0275
0.128
Peak1
2.025
2.547 1.9629215 0.5452541 1.723437 0.7840714
LSSD2
100
100 N/A
N/A
3.7 N/A
Relative Luciferase Activity
3
TR
2
1
PFOS PFOS217 PFHxS PFBS PFOA PFBA
Relative Luciferase Activity
0
-1
0
1
2
Log(Dose) M
4
3 PXR
2
1
PFOS PFOS217 PFHxS PFBS PFOA PFBA
0
-1
0
1
2
Log(Dose) M
4
3
CAR3
2
1
PFOS PFOS217 PFHxS PFBS PFOA PFBA
0
-1
0
1
2
Log(Dose) M
Relative Luciferase Activity