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Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation of MCF-7 breast cancer cells, and cotreatment with the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 and the antiestrogen ICI 182780 inhibits IGF-I-induced growth. The role of estrogen receptor α (ERα) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ERα (iERα). The results showed that IGF-I-dependent phosphorylation of Akt and mitogen-activated protein kinase, induction of G1–S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ERα. Moreover, these same IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780 and this was in contrast to a previous report suggesting that ICI 182780 did not affect IGF-I-dependent activation of PI3-K or induction of cyclin D1 expression. ICI 182780 exhibits antimitogenic activity and iERα inhibits G1–S-phase progression and proliferation of MCF-7 cells treated with IGF-I, suggesting that the effects of the antiestrogen are primarily related to downregulation of ERα.

Wildlife and humans are exposed to a complex mixture of endocrine active chemicals. The activity of a specific chemical in any mixture can be modified through interactions with other components of the mixture. The toxic equivalency factor (TEF) approach for risk assessment was developed for chemicals such as halogenated aromatics that induce their effects through ligand-activated receptors. For persistent halogenated aromatic AhR agonists, this approach has some utility. However, the use of the TEF approach for endocrine active compounds is confounded by the unique tissue- and response-specific activities of these structurally diverse compounds. The term selective receptor modulator describes the ability of a natural or synthetic receptor ligand to manifest agonist activity in one tissue or for one response and antagonist activity in other tissues or for another response in the same tissue. Thus, it is possible for chemicals in a mixture to behave in an additive manner for one response and an antagonist manner for another response. A mechanisms-based hazard risk assessment of endocrine active chemical mixtures must account for these multiple variables.

Transferrin (Tf) is an iron transport protein expressed in MCF-7 human breast cancer cells. In nuclear run-on assays, 17beta-estradiol (E2) increased the rate of Tf gene expression approximately 3-fold within 1 h after treatment and reporter gene activity was also induced in MCF-7 cells transfected with a construct containing a -3600 to +39 Tf gene promoter insert. Deletion and mutation analysis identified an E2-responsive promoter region between -811 and -762, which was GC-rich (80%) and contained two nonconsensus estrogen response elements (EREs). E2-responsiveness of this region was associated with a GGACA(N)(3)TGGCC motif (-803 to -791) which bound human estrogen receptor alpha (hERalpha) in gel mobility shift assays. In Drosophila Schneider SL-2 cells, the -811 to -752 was E2-responsive after cotransfection with hERalpha expression plasmid plus E2, whereas Sp1 protein did not induce transactivation. These studies confirm that E2 induces Tf gene expression through a nonconsensus distal ERE.

Cyclin D1 gene expression is induced by 17beta-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G(1) phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen- and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at -66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GC-rich Sp1-binding sites at -143 to -110 were also E2-responsive, and interaction of ERalpha and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ERalpha/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.

Aryl hydrocarbon receptor (AhR) agonists inhibit 17β-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)+RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 μM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response α, EGRα) and other proteins involved in signaling pathways (calmodulin, ATP synthase α subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.

The cytochrome P450 (CYP) gene products such as CYP3A and CYP2B are essential for the metabolism of steroid hormones and xenochemicals including prescription drugs. Nuclear receptor SXR/PXR (steroid and xenobiotic receptor/pregnenolone X receptor) has been shown both biochemically and genetically to activate CYP3A genes, while similar studies have established constitutive androstane receptor (CAR) as a CYP2B regulator. The response elements in these genes are also distinct, furthering the concept of independent regulation. Unexpectedly, we found that SXR can regulate CYP2B, both in cultured cells and in transgenic mice via adaptive recognition of the phenobarbital response element (PBRE). In a type of functional symmetry, orphan receptor CAR was also found to activate CYP3A through previously defined SXR/PXR response elements. These observations not only provide a rational explanation for the activation of multiple CYP gene classes by certain xenobiotics, but also reveal the existence of a metabolic safety net that confers a second layer of protection to the harmful effects of toxic compounds and at the same time increases the propensity for drug-drug interactions.

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

17beta-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter elements in which Sp1 but not ER binds DNA. This study reports the ligand- and cell context-dependent ER(alpha)/Sp1 and ER(beta)/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER(alpha) and ER(beta) proteins physically interact with Sp1 (coimmunoprecipitation) and preferentially bind to the C-terminal region of this protein in pull-down assays. E2- and antiestrogen-dependent transcriptional activation of ER(alpha)/Sp1 was observed in MCF-7, MDA-MB-231, and LnCaP cells, but not in HeLa cells. E2 did not affect or significantly decrease ER(beta)/Sp1 action, and antiestrogens had minimal effects in the same 4 cell lines. Exchange of activation function-1 (AF-1) domains of ER subtypes gave chimeric ER(alpha/beta) (AF-1alpha/AF-2beta) and ER(beta/alpha) (AF-1beta/AF-2alpha) proteins that resembled wild-type ER (alpha or beta) in terms of physical association with Sp1 protein. Transcriptional activation studies with chimeric ER(beta/alpha) and ER(alpha/beta) showed that only ER(alpha/beta) can activate transcription from an Sp1 element, not ER(beta/alpha). This indicates that the AF-1 domain from ER(alpha) is responsible for activation at an Sp1 element, independent of ER subtype context. In order to further characterize this observation, deletion constructs in the AF-1 domain of both ER(alpha) and ER(alpha/beta) were made, and transactivation studies indicated that the region between amino acids 79 and 117 of this domain is important for activation at an Sp1 element.

bcl-2 gene expression is induced by 17beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ER(alpha)) did not bind [(32)P]bcl-2j, whereas Sp1 protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Sp1 protein. Subsequent studies confirmed that transactivation by E2 was dependent on ER(alpha)/Sp1 interactions with both GC-rich sites, and this was confirmed by in vitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Sp1 or ER(alpha) protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif. These data coupled with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element. Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.

3,3′,4,4′-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3′,4,4′-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3′,4,4′tetraCB. Our results showed that 3,3′,4,4′-tetraCB and a structurally related analog, 3,3′,4,4′,5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3′,4,4′-tetraCB and 3,3′,4,4′,5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21-25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17β-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3′,4,4′-tetraCB significantly inhibited mammary tumor growth in female Sprague-Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3′,4,4′-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR-ER crosstalk.

3,3',4,4'-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3',4,4'-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3',4,4'-tetraCB. Our results showed that 3,3',4,4'tetraCB and a structurally related analog, 3,3',4,4',5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21-25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17beta-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3',4,4'-tetraCB significantly inhibited mammary tumor growth in female Sprague-Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3',4,4'-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR-ER crosstalk.

The role of positive co-operativity in stabilizing the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the rat hepatic cytosolic TCDD receptor protein (Ah receptor) was investigated. The binding mechanism of TCDD was determined by kinetic means through equilibrium and saturation binding studies, and Scatchard and Hill plot analysis. In all studies, the slope of the Hill plot was close to 1.0, indicating the absence of positive co-operativity. Interpretation of the Scatchard plot was however complicated by the fact that both linear and nonlinear plots were experimentally obtained. The nonlinearity was shown to be an experimental artifact and a consequence not of co-operativity, but of high levels of nonspecific binding. The high level of nonspecific binding could be attributed to: (1) lipophilicity of the TCDD ligand, and (2) inefficient competition of receptor-bound [3H]TCDD. When nonspecific binding was minimized, the Scatchard slope was linear and in agreement with the Hill coefficient, thus indicating the lack of positive co-operativity in the binding of TCDD to the Ah receptor.

The identification of the hydroxylated rat urinary metabolites of the 2-, 3- and 4-chlorobiphenyls and 2-, 3- and 4-bromobiphenyls has been determined by gas chromatographic mass spectrometric analysis of their corresponding methyl ether derivatives. The electron impact fragmentation patterns of the bromomethoxybiphenyls and chloromethoxybiphenyls were used to assign the position of the methoxyl group (ortho, metaor parato the biphenyl bond); the mass spectra of the corresponding [2H5]halobiphenyls confirmed the sites of hydroxylation by distinguishing between the halophenyl and phenyl rings. The results illustrated that ring hydroxylation occurs predominantly at the parapositions of the biphenyl nucleus and at sites which are orthoand parato the halogen substituents. 4,4'-Dimethoxyhalobiphenyls are major urinary metabolites of the 2- and 3-halobiphenyls and the rapid formation of these metabolites is illustrated in a time course study which monitors the urinary metabolites formed after the separate coadministration of the isomeric chlorobiphenyl and bromobiphenyl substrates to rats.

This paper reports the development and application of an analytical method in which both polyethylene glycol ethers or polyethylene glycols are converted into ethylene dibromide via treatment with concentrated hydrobromic acid/glacial acetic acid (1:1) at 150°C for 3 hours. This method can detect <1 ppm of the surfactant in biological tissues, however, the practical utility of the technique is dependent on the solvent and reagent purities and the various cleanup methods which include base hydrolysis, high pressure liquid chromatography and Florisil or acidified Florisil column chromatography.

Hazard and risk assessment of individual toxic chemicals utilizes data obtained from chronic toxicity and carcinogenicity studies in laboratory animals for regulating emission, cleanup or intake levels for individual chemicals. This approach can be utilized for problems associated with a single chemical emitted from a point source; however, in most situations, toxic chemicals are formed and emitted into the environment as complex mixtures of different structural classes of toxic/carcinogenic chemicals. Methodologies for risk assessment of complex mixtures have been developed for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) which are found as industrial and combustion by-products. This methodology is based on the common mechanism of action for these chemicals and utilizes the most toxic PCDD/PCDF congener, 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as a reference compound. The relative potencies or toxic equivalency factors (TEFs) are assigned to the other relevant PCDD/PCDF congeners and the TEF = EC50 or ED50(TCDD)/EC50 or ED50 (test congener). Thus, for any complex mixture of PCDDs and PCDFs, the TCDD-like toxicity or toxic equivalents (TEQ) for the mixture can be calculated using the equation: TEQ = S [TEFi × PCDDi]n × S [TEFi × PCDFi]n where TEFi is the TEF for the individual PCDD or PCDF congener and n is the number of compounds in each mixture. The TEF approach is currently used by regulatory agencies for risk management of PCDDs and PCDFs as well as being considered for polychlorinated biphenyls (PCBs). This paper discusses the problems associated with the TEF approach for risk assessment of halogenated aromatic and polycyclic aromatic hydrocarbons.

Chlorinated trans stilbenes bind with high affinity to the cytosolic Ah receptor from Wistar rat liver. The EC50 values for competition with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cover a range from 10-400 nM, but with no apparent relationship between molecular structure and binding activity. These compounds induce monooxygenase enzyme activity in rat hepatoma H-4-II E cells in culture only weakly, again with no apparent dependence on molecular structure, and the weakest enzyme inducers act as antagonists for the induction of these enzymes by TCDD. In vivo administration of 3,3',4,4'-tetrachloro-tans-stilbene in the rat leads to decreased Ah receptor levels.

Hexachlorobenzene (400 μimol/kg) induced rat hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD), however at dose levels up to 3000 μmol/kg, the induced enzyme levels were less than 50% of the maximum induced activities caused by either 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). HCB did not significantly induce these monooxygenases in the thymus or lung but some time-dependent induction of AHH and EROD was observed in the rat kidney. In contrast, MC induced microsomal AHH and EROD in the rat liver, lung and kidney but not the thymus. Cotreatment of HCB (400 μmol/kg) plus 2,3,7,8-TCDD (10 or 30 μg/kg) resulted in some small but significant dose- and organ-dependent modulation of the activity of 2,3,7,8-TCDD as an inducer of AHH and EROD. The 30 μg/kg dose of 2,3,7,8-TCDD was toxic and this may account for the lower induced enzyme levels observed in the kidney and thymus of animals cotreated with HCB plus 2,3,7,8-TCDD compared to rats given 2,3,7,8-TCDD alone. Animals cotreated with 2,3,7,8-TCDD (10 μg/kg) plus HCB exhibited decreased AHH and EROD activities in the liver and thymus but increased enzyme levels in the kidney. In a separate study, it was shown that over a 14-day period, HCB caused significant decreases in both the lung and kidney aryl hydrocarbon (Ah) receptor binding levels but no significant long-term changes in hepatic or thymic receptor levels. The role of the HCB-mediated changes in receptor binding levels and its relationship to the induction of AHH and EROD by 2,3,7,8-TCDD is unclear. The most dramatic interactive effects of HCB (400 μmol/kg) and 2,3,7,8-TCDD (10 or 30 μg/kg) were observed with two Ah receptor-mediated toxic effects, namely body weight loss and thymic atrophy. HCB synergistically exacerbated both body weight loss and thymic atrophy caused by 2,3,7,8-TCDD whereas HCB administered at doses as high as 3000 μmol/kg did not cause any significant body weight loss or thymic atrophy in the rats. The mechanism of these toxic interactions are unknown.

Several hydroxylated polychlorinated biphenyls (PCBs) identified in human serum have been synthesized and these include 2,2′,3,4′,5,5′-hexachloro-4-biphenylol; 2,3,3′,4′,5-pentachloro-4-biphenylol; 2′,3,3′,4′,5-pentachloro-4-biphenylol; 2,2′,3,3′,4′,5-hexachloro-4-biphenylol; 2,2′,3,3′,4′,5,5′-heptachloro-4-biphenylol; 2,2′,3,4′,5,5′,6-heptachloro-4-biphenylol; and 2,2′,3′,4,4′,5,5′-heptachloro-3-biphenylol. The hydroxy-PCBs exhibited minimal binding to the rat uterine cytosolic estrogen receptor (ER) and did not induce proliferation of estrogen-responsive MCF-7 human breast cancer cells at concentrations ranging from 10−5to 10−8m. The estrogenic activity of these compounds was further investigated utilizing two estrogen-responsivein vitrobioassays, namely, (i) HeLa cells stably transfected with a Gal4:human ER chimera and a 17-mer-regulated luciferase reporter gene, and (ii) MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene. None of the hydroxy-PCBs significantly induced luciferase activity in the stably transfected HeLa cells or CAT activity in MCF-7 cells at concentrations as high as 10−5m. The antiestrogenic effects of the hydroxy-PCBs were also investigated using the same bioassays in which the cells were cotreated with 17β-estradiol plus the hydroxy-PCBs. All of the hydroxy-PCB congeners inhibited one or more estrogenic response, and one congener, 2,2′,3,4′,5,5′,6-heptachloro-4-biphenylol, inhibited 17β-estradiol-induced cell proliferation and CAT activity in MCF-7 cells and luciferase activity in HeLa cells.

Wild-type Neurospora crassa is completely inhibited by 5 ppm nystatin. Ultraviolet-induced mutants have been isolated that grow in the presence of 60 ppm of the antibiotic. Gas-liquid chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses showed the wild-type sterols to be ergosterol (ergosta-5,7,22-trien-3ß-ol) and episterol (ergosta-7,24(28)-dien-3ß-ol) in a 3:1 ratio. The mutants contained lichesterol (ergosta-5,8,22-trien-3ß-ol) and fecosterol (ergosta-8,24(28)-dien-3ß-ol) in a 2:1 ratio, differing from the wild type only in the position of the B-ring unsaturation. A deficiency of an ergosta-8,24 (28)-dien-3ß-ol:ergosta-7,24(28)-dien-3ß-ol isomerase is indicated.

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and previous analyses of the proximal promoter region of this gene identified two functional enhancer sequences; namely an Sp1(N)23estrogen-responsive element (ERE) half-site (-199 to -165) and an imperfect palindromic ERE (-119 to -107). A third region of the cathepsin D gene promoter (CD/L, -145 to -119) was also E2 responsive in transient transfection assays. A GC-rich sequence which contains two overlapping Sp1 binding sites (-145 to -135) was responsible for ER-mediated transactivation and required formation of an ER/Sp1 complex in which only the Sp1 protein bound DNA. E2 responsiveness of the CD/L sequence was also dependent on an adjacent overlapping GCGTG motif corresponding to the dioxin-responsive element (DRE) core binding sequence, which is the cognate response element for the heterodimeric aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) transcription factor complex. The results show that ER-mediated transactivation of CD/L was associated with the Sp1(N)2-4DRE (core) motif and involved formation of a multiprotein ER/Sp1-AhR/ARNT complex. These results illustrate a unique example of an endogenous role for AhR/ARNT in the absence of added AhR agonist and indicate that the cathepsin D gene proximal promoter region contains at least three different functional motifs associated with ER-mediated transactivation.

alpha-Naphthoflavone (alpha NF) is a weak aryl hydrocarbon (Ah) receptor agonist and inhibits the induction of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. It has been suggested that the Ah receptor antagonist activity is due to the formation of alpha NF-cytosolic Ah receptor complexes that fail to undergo transformation. This hypothesis is consistent with data obtained in this and other studies using alpha NF concentrations from 10 to 1000 nM. However, 10 microM alpha NF exhibited Ah receptor agonist activity in several assays. Incubation of rat hepatic cytosol with 10 microM alpha NF caused transformation of the Ah receptor, as determined in a gel retardation assay using a 32P-labeled oligonucleotide containing a single dioxin-responsive element (DRE). Incubation of rat hepatoma (H-4-II E) cells with 10 microM alpha NF not only resulted in the induction of CYP1A1 mRNA levels but also increased chloramphenicol acetyltransferase activity from a DRE-containing chloramphenicol acetyltransferase reporter plasmid. Moreover, the DRE-transformed cytosolic Ah receptor complex liganded with either alpha NF or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not undergo significant dissociation at 4 degrees. These data confirm that alpha NF is an Ah receptor agonist and, based on the results of previous studies, exhibits partial antagonist activity via competition for receptor binding sites.

Abstract: Purpose: 1,3,6,8-Substituted alkyl polychlorinated dibenzofurans (PCDFs), typified by 6-methyl-1,3,8-triCDF (MCDF), inhibit 17β-estradiol (E2)-induced responses in the rodent uterus and human breast cancer cells. The major purpose of the experiments reported here was to determine the structure-dependent antiestrogenic activities of several alternate-substituted (1,3,6,8- and 2,4,6,8-) PCDFs. Methods: The antiestrogenic activities were determined in MCF-7 human breast cancer cells using two assays, that is E2-induced cell proliferation and induction of chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with the E2-responsive Vit-CAT plasmid. Results: MCDF (10−5 M ), 6-isopropyl-1,3,8-triCDF, 6-ethyl-1,3,8-triCDF, 3-isopropyl-6-methyl-1,8-diCDF, and 6-methyl-2,4,8-triCDF, inhibited both E2-induced cell proliferation and CAT activity in MCF-7 cells. All of the remaining ten congeners inhibited either E2-induced cell proliferation or CAT activity, but not both responses. Conclusions: The antiestrogenic activity of the alternate-substituted PCDFs involves interactions between the aryl hydrocarbon and estrogen receptor signaling pathways. Although these compounds exhibited antiestrogenic activity in MCF-7 cells, the effects of individual congeners were response-specific, and there were no apparent structure-activity relationships.

The photolysis products and reaction quantum yields from a number of simple halonaphthalenes have been determined. Fluoronaphthalenes react from a singlet state to yield substitution products in nucleophilic solvents. Chloronaphthalenes and 1-bromonaphthalene afford mainly radical products (naphthalene and binaphthyls) through a triplet state, though some photonucleophilic substitution occurs with chloronaphthalene. Attempts at quenching lead to abnormal behavior, in that enhanced quantum yields for reaction are found with several potential triplet quenchers. Electron transfer processes are proposed to rationalize the observed products.

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) binds with moderate affinity to the aryl hydrocarbon (Ah) receptor protein (4.9 x 10(-8) M) but is a weak Ah receptor agonist. Cotreatment of male Long Evans rats with MCDF (50 mumol/kg) and a dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that causes a near-maximal induction of hepatic microsomal aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase activities resulted in a significant inhibition of these activities for up to 96 hr. Comparable results were obtained with MCDF (10(-7) M) and TCDD (10(-8) M) in rat hepatoma H-4-II E cells in culture over 36 hr. TCDD treatment of rats resulted in an initial decrease of hepatic cytosolic Ah receptor within 6 hr and this was followed by a subsequent 138% increase in cytosolic receptor levels 72 hr after treatment. Although MCDF (50 mumol/kg) did not significantly alter rat hepatic cytosolic Ah receptor levels in animals cotreated with TCDD plus MCDF, the latter compound significantly inhibited TCDD-mediated replenishment of the cytosolic Ah receptor. In contrast, treatment of rat hepatoma H-4-II E cells with TCDD (10(-8) M) resulted in the rapid (within 1 hr) depletion of cytosolic Ah receptor, which remained undetectable for up to 36 hr; cotreatment of the cells with MCDF (10(-7) M) and TCDD (10(-8) M) resulted in cytosolic Ah receptor levels that were similar to those observed after treatment with TCDD alone. The effects of MCDF on the uptake and persistence of nuclear [3H]TCDD-Ah receptor complex levels were also determined in rat liver and rat hepatoma H-4-II E cells in culture. MCDF did not significantly decrease levels of occupied nuclear Ah receptor complexes in the rat or rat hepatoma cells. Moreover, using the sucrose density gradient assay procedure, the sedimentation coefficients of the cytosolic and nuclear TCDD-Ah receptor complexes in the presence or absence of MCDF were comparable. The results of these and other related studies with 6-substituted-1,3,8-trichlorodibenzofurans suggest that MCDF may act as a partial TCDD antagonist by competing with TCDD for nuclear binding sites.

The binding affinities of 16 7-substituted 2,3-dichlorodibenzo-p-dioxins for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein from male Wistar rats have been determined. The EC50 value for each compound was estimated by competitive displacement of [3H]TCDD and the data illustrated that the differences between competitive ligands were dependent on the substituent (X) group. The EC50 value for 7-trifluoromethyl-2,3-dichlorodibenzo-p-dioxin was 1.95 X 10(-8) M and was greater than 1000-fold more active than 7-amino-2,3-dichlorodibenzo-p-dioxin (EC50 = 2.88 X 10(-5) M). Multiple parameter linear regression analysis of the data for 14 different compounds gave the following equation: log (1/EC50) = 1.24 pi + 6.11. This demonstrated that the binding affinity was linearly dependent on the lipophilicity (pi) of the 7-X-group. This contrasted with a comparable analysis of the substituent effects on the binding of 13 4'-substituted 2,3,4,5-tetrachlorobiphenyls to the cytosolic receptor which showed that the lipophilicity, electronegativity, and hydrogen-bonding capacity were important physicochemical determinants which facilitated binding to the receptor protein. These data suggest that the halogenated dibenzo-p-dioxins and biphenyls may interact with different binding sites on the receptor or they may bind to the same site but exert different conformational effects on the receptor protein. For the 7-X-2,3,-dichlorodibenzo-p-dioxins, there was not a rank order correlation between receptor-binding EC50 values and the induction of aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase in rat hepatoma H-4-II E cells in culture. However, the data could be correlated with an estimate of substituent width, the STERIMOL factor (B5), i.e., log (AHH) = 1.29 log (binding) + 2.19 delta B5 - 1.31 (delta B5)2 - 1.48. The importance of a steric factor in the correlation between receptor binding and AHH induction for substituted dibenzo-p-dioxins and halogenated biphenyls is consistent with a structure-dependent conformational change(s) in the receptor protein:ligand complex after the initial binding event. Presumably, this latter process is associated with the steps involving interactions between the ligand:receptor complex and nuclear binding sites.

Immunosuppressive Activity of Polychlorinated Biphenyl Mixtures and Congeners: Nonadditive (Antagonistic) Interactions. Harper, N., Connor, K., Steinberg, M., and Safe, S. (1995). Fundam. Appl. Toxicol. 27, 131-139. The dose-response inhibition of the splenic plaque-forming cell (PFC) response and serum IgM units to the antigen, trinitrophenyl-lipopolysaccharide, was determined for several polychlorinated biphenyl (PCB) mixtures and congeners in female B3C3F1 mice. The ED50 values for Aroclor 1260-, 1254-, 1248-, and 1242-induced immunotoxicity varied by less than twofold from 355 to 699 mg/kg. The range of ED50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5-pentaCB, 3,3',4,4',5,5'-hexaCB, 2,3,3',4,4'-pentaCB, 2,3',4,4',5-pentaCB, 2,3,3',4,4',5-hexaCB, 2,3,3',4,4',5,5'-heptaCB, 2,2',3,3',4,4',5-heptaCB, and 2,2',3,4,4',5,5'-heptaCB were 4.6 to 4.9, 134 to 245, 4.7 to 7.0, 6.9 to 11.1, 88,000 to 121,000, 122,000 to 132,000, 99,000 to 157,000, 89,000 to 129,000, 117,000 to 240,000, and 132,000 to 238,000 µg/kg, respectively. The immunotoxicity-derived toxic equivalency factors (TEFs) for these congeners could be calculated from the ED50 (TCDD)/ED50 (congener) ratios and the TEF values were within the range of those previously determined for other aryl hydrocarbon receptor-mediated responses. Based on the known concentrations of these congeners in the PCB mixtures, TCDD or toxic equivalents (TEQs) in the mixture were calculated [i.e., TEQ = Σ (PCB_congener × TEF)] using the immunotoxicity-derived TEFs (plaque-forming cells/10⁶ viable cells). TEQ values for Aroclors 1260, 1254, 1248, and 1242 were 16.0, 54.4, 260.4, and 197 ppm, respectively. Based on the ED50 value for the immunosuppressive activity of TCDD (4.8 µg/kg), the calculated ED50 values for immune suppression by Aroclors 1260, 1254, 1248, and 1242 were 300, 88, 18, and 24 mg/kg, respectively. The ED50 (observed)/ED50 (calculated) ratios were 1.2, 5.9, 21, and 22.0 for Aroclors 1260, 1254, 1248 and 1242, respectively. Thus, for Aroclors 1254, 1248, and 1242, the high ED50 (observed)/ED50 (calculated) ratios (i.e., 5.9 to 22.0) indicate that the TEF approach overestimates the toxicity of these mixtures due to nonadditive (antagonistic) interactions of the PCBs. In contrast, the TEF approach was useful in determining the immunotoxicity of the Aroclor 1260 mixture. Copyright 1995, 1999 Academic Press

The effects of structure on the estrogenicity and antiestrogenicity of hydroxylated polychlorinated biphenyls were investigated using the following estrogen-sensitive assays: competitive binding to the rat and mouse cytosolic estrogen receptor (ER); immature rat and mouse uterine wet weight, peroxidase and progesterone receptor (PR) levels; induction of luciferase activity in HeLa cells stably transfected with a Gal4:human ER chimera and a 17mer-regulated luciferase reporter gene; proliferation of MCF-7 human breast cancer cells; induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a CAT reporter gene. The chemicals synthesized for this study contained a 4-hydroxy group in one ring, a 2- or 3-chloro substituentmetaororthoto the hydroxyl group, and variable substitution (2′,3′,4′,5′-, 2′,3′,4′,6′-, 2′,3′,5′,6′-tetrachloro and 2′,4′,6′-trichloro) in the chlorophenyl ring. The compounds included: 2,2′,3′,4′,5′- (A), 2,2′,3′,4′,6′- (B), and 2,2′,3′,5′,6′-pentachloro- (C); 2,2′,4′,6′-tetrachloro-4-biphenylol (D); 2′,3,3′,4′,5′- (E), 2′,3,3′,4′,6′- (F), and 2′,3,3′,5′,6′-pentachloro (G); and 2′,3,4′,6′-tetrachloro-4-biphenylol (H). With the exception of 2′,3,4′,6′-tetrachloro-4-biphenylol (H), all of the compounds competitively bound to the mouse and rat ER with relative binding affinities [compared to 17β-estradiol (E2)] varying from 1.4 × 10−3to 5.3 × 10−5. The structure–ER binding relationships for the hydroxy-PCB congeners were different in the rat and mouse, and no dose-dependent estrogenic activities were observed in the mouse or rat uterus. Several hydroxy-PCB congeners exhibited antiestrogenic activity (primarily in the mouse uterus) and two compounds, 2,2′,3′,5′,6- and 2,2′,3′,4′,6′-pentachloro-4-biphenylol, inhibited E2-induced uterine wet weight, PR binding, and peroxidase activity in the mouse uterus. 2,2′,3′,4′,5′- and 2,2′,3′,4′,6′-Pentachloro-4-biphenylol induced CAT activity in MCF-7 cells transiently transfected with the Vit-CAT plasmid; the remaining congeners did not induce CAT activity but exhibited antiestrogenic activity in MCF-7 cells cotreated with 10−9E2 plus 10−5mhydroxy-PCBs. Complementary structure–estrogenicity relationships were observed utilizing the HeLa cell luciferase induction and MCF-7 cell proliferation assays. The placement of the 2- or 3-chloro groups in the phenolic ring had minimal effects on estrogenic activity, whereas 2,4,6-trichloro- and 2,3,4,6-tetrachloro substitution in the chlorophenyl ring (B, D, F, and H) were required for this response. Substitution in the phenolic ring was also not important for structure–antiestrogenicity relationships, and the most active compounds (A, C, E, and G) contained 2′,3′,4′,5′- and 2′,3′,5′,6′-tetrachlorophenyl groups. Thus, structure–estrogenicity/antiestrogenicity relationships for this series of hydroxy-PCBs were complex and response-specific.

α-Naphthotlavone (αNF) and 6-methyl-1,3,8-trichlorodiben-zofuran (MCDF) inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1gene expression in MCF-7 human breast cancer cells and also decreased the accumulation of the nuclear [3H]TCDD-aryl hydrocarbon (Ah) receptor complex. Nuclear extracts from cells treated with 10−6M αNF and incubated with a dioxin responsive element (DRE, 26-mer) did not form a retarded band in a gel mobility shift assay. In contrast, incubation of nuclear extracts from cells treated with 10−6M MCDF and DRE gave a retarded band and this is consistent with the antiestrogenic and Ah receptor agonist activity of MCDF in human breast cancer cells. αNF was further investigated as an Ah receptor antagonist by determining the inhibition by αNF of TCDD-induced antiestrogenicity in MCF-7 cells. TCDD (10−9M) inhibited the 17β-estradiol-induced proliferation of MCF-7 cells and the secretion of the 52-kDa protein. In cotreatment studies, αNF (10−8to 10−6M) caused a concentration-dependent decrease in the antiestrogenic responses elicited by TCDD. In addition, αNF inhibited the TCDD-induced down-regulation of nuclear estrogen receptor levels in MCF-7 cells. αNF (10−6 M) alone was inactive as an estrogen or antiestrogen and in cotreatment studies did not affect 17β-estradiol-induced responses in MCF-7 cells. Tamoxifen (10−7M), an antiestrogen which acts through the estrogen receptor, also inhibited 17β-estradiol-induced cell proliferation and aNF did not affect the tamoxifen-mediated antiproliferative response. Thus, aNF antagonized TCDD-induced CYP1A1gene expression in MCF-7 cells and also acted as an anti-antiestrogen for TCDDmediated antiestrogemcity in these cells. These results were consistent with the low levels of DRE binding observed with nuclear extracts from cells treated with 10−9M TCDD plus αNF (10−8to 10−6M) or 10−6M αNF alone. Thus, αNF appears to act as an Ah receptor antagonist in MCF-7 cells by decreasing the levels of transcriptionally active nuclear Ah receptor complexes.

Abstract:  Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. 32P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with 32P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6�106 DNA nucleotides in skin (both extracts) and one adduct in 3.2�107 or 1.2�107 DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the 32P-labeled derivative of the reaction product of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N 2 of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by 32P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts.

The effects of o-, m- and p-terphenyl, 2,4-dichloro-, 2,4,6-trichloro-, 2,3,5,6-tetrachloro-, 2,3,4,6-tetrachloro-, 2,4,4'",6- tetrachloro- and 2,3,4,5-tetrachloro-p-terphenyl, 2,3,4,5-tetrachloro-m- and o-terphenyl as inducers of hepatic drug-metabolizing enzymes were determined in immature male Wistar rats. o-Terphenyl, 2,4-dichloro-, 2,4,6-trichloro-p-terphenyl and 2,3,4,5-tetrachloro-o-terphenyl induced 4,4'-dimethylamino antipyrine N-demethylase at total dose levels of 300 µmol/kg and the 2,3,4,5-tetrachloro-p-terphenyl induced ethoxyresorufin O-deethylase (EROD). In contrast, none of the other terphenyls or polychlorinated terphenyls (PCTs) induced these enzyme activities. Previous studies have demonstrated that 2,3,4,5-tetrachloro-p-terphenyl did not exhibit a high affinity for the 2,3,7,8-tetrachlorodibenzo-p-trachlorodibenzo-p-dioxin (TCDD) receptor protein (EC50= 6.6×10-6M). In contrast, this study showed that 2,3,4,5-tetrachloro-p-terphenyl was more active than either 2,3,4,5-tetrachloro-o- or m-terphenyl as an inducer of EROD. Moreover, the competitive receptor binding EC50 values for the latter two isomers were > 10-5 M and this result was also consistent with their lack of EROD induction activity. Previous studies showed that analysis of the data for a series of 4'-substituted-2,3,4,5-tetrachlorobiphenyls indicated that the p-terphenyl structural moiety (i.e. 4'-substituent = phenyl) did not interact with high affinity with the receptor protein binding site. Since the 2,3,4,5-tetrachloro o- and m-terphenyls are also poor ligands for the receptor protein, this data and results from other studies indicate that PCT congeners (and commercial mixtures) are therefore unlikely to elicit significant 2,3,7,8-TCDD-like biologic or toxic effects in target species.

The estrogen receptor and aryl hydrocarbon receptor (AhR) are coexpressed in several Ah and estrogen-responsive human breast cancer cell lines. However, a recent study reported that 17beta-estradiol (E2) inhibited Ah responsiveness in mouse Hepa 1c1c7 hepatoma cells (Kharat, I., and Saatcioglu, F. (1996) J. Biol. Chem. 271, 10533-10537), and therefore, estrogen receptor-AhR cross-talk was reinvestigated in MCF-7 and mouse Hepa 1c1c7 cells. Treatment of MCF-7 or Hepa 1c1c7 cells with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in induction of CYP1A1-dependent activity and mRNA levels. Treatment of both cell lines with E2 had no effect on basal or TCDD-inducible CYP1A1-dependent activity or mRNA levels. In MCF-7 and Hepa 1c1c7 cells transiently transfected with an Ah-responsive plasmid containing the 5'-regulatory region of the human CYP1A1 gene fused to the chloramphenicol acetyltransferase reporter gene 10 nM TCDD significantly induced chloramphenicol acetyltransferase activity; in cells cotreated with TCDD plus E2 the induced response was not affected by the hormone. Nuclear extracts from cells treated with dimethyl sulfoxide, E2, TCDD, and TCDD plus E2 were incubated with the [32P]dioxin-responsive element and analyzed by gel electrophoretic mobility shift assays. A retarded band associated with formation of a [32P]dioxin-responsive element-AhR complex was observed in nuclear extracts from cells treated with TCDD or TCDD plus E2 (cotreated). Collectively these studies suggest that E2 does not modulate AhR-mediated CYP1A1 gene expression in MCF-7 or Hepa 1c1c7 cells.