Prolactin-stimulated polymerization of acetyl-coA carboxylase in explants of mid-pregnant rat mammary gland.

Prolactin significantly increased the rate of fatty acid synthesis in explants of mid-pregnant rat mammary gland cultured for 96 h with insulin plus corticosterone. Under these conditions, prolactin increased the specific activity of total acetyl-CoA carboxylase in nuclear-free homogenates of explants by 2.6, and increased the proportion of the enzyme in the active polymeric form from 0.44 to 0.89. Removal of prolactin after 48 h in culture decreased the specific activity of the total enzyme by about half. and decreased the proportion as polymer to 0.52. The results show that prolactin plays a major role in mid-pregnant rat mammary gland in the polymerization which accompanies increased activity of the total enzyme and increased rate of fatty acid synthesis.

3,4,3',4'-Tetrachlorobiphenyl acts as an estrogen in vitro and in vivo.

Polychlorinated biphenyls (PCBs) are one of the most widespread, persistent man-made products in the ecosystem giving rise to serious environmental contamination and potential hazard to health. The PCBs, in common with other compounds such as the dioxins, have been shown to exert some biological actions mediated through the aryl hydrocarbon receptor. Evidence for interaction of PCBs with other nuclear receptors has been sparse. Here we present evidence that 3,4,3',4'-tetrachlorobiphenyl (TCB) (PCB77), a PCB with high toxicity and significant bioaccumulation, can act as an estrogen with actions mediated through the estrogen receptor. Evidence is presented from multiple assay systems including 1) ligand binding to estrogen receptor in a competitive binding assay, 2) ligand ability to induce estrogen receptor binding to DNA, 3) ligand regulation of gene expression from a transfected exogenous (ERE-tk-CAT) or an endogenous (pS2) estrogen-regulated gene, 4) ligand regulation of cell growth in estrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1, and 5) ligand activity in the immature mouse uterine weight bioassay in vivo. These results demonstrate that TCB (PCB77) can be included in the increasing list of environmental pollutants that possess the ability to mimic estrogen action and be termed an environmental estrogen. Since the concentrations of TCB used here (10(-9) M; 292 ng/liter) are not incompatible with levels of PCB/TCB found in human tissues, these results may have physiological relevance. Use of multiple approaches to study estrogenic action demonstrates that one congener can act as both an agonist and antagonist of estrogen action and that the magnitude of these effects can alter according to the molecular environment.

Milk whey proteins in plasma of sows: variation with physiological state.

The whey proteins alpha-lactalbumin and beta-lactoglobulin have been investigated as potential markers of mammary development in sows by measuring their concentrations in plasma. The whey proteins were isolated from porcine milk by gel filtration, ion-exchange and hydrophobic interaction chromatography, characterized by several criteria and used to raise antibodies. Specific radioimmunoassays were set up for porcine alpha-lactalbumin and beta-lactoglobulin and validated for use in porcine blood and milk. Plasma levels of the whey proteins were measured in sows that were pregnant, suckling litters post partum, weaned abruptly at birth or were pregnant but mastectomized. Both whey proteins showed similar patterns in plasma post partum, falling from a maximum 1 d after parturition to values < 0.02% those in milk by day 4-5 post partum in suckling sows and showing a transient peak associated with early involution before declining to very low concentrations in non-suckling sows. alpha-Lactalbumin was first detected in the last week prepartum, rising markedly in the 3 d before parturition, correlated with rising prolactin (r = 0.986) and falling progesterone (r = -0.998). beta-Lactoglobulin rose much earlier from 5 weeks prepartum, at the time when lobulo-alveolar mammary development is occurring, and correlated (r = 0.929) with oestradiol-17 beta. In mastectomized sows, concentrations of whey proteins in plasma were reduced by 90% or more when compared with intact animals, though following a similar pattern. This study shows that whey protein concentrations in plasma vary with physiological state and reflect aspects of the development of the mammary gland. The very different profiles for alpha-lactalbumin and beta-lactoglobulin prepartum indicate that they are differently controlled.

Hormonal induction of alpha-lactalbumin and beta-lactoglobulin in cultured mammary explants from pregnant pigs.

Mammary tissue from pigs on days 60, 80, 90, 100 and 100+ (days 106-111) of pregnancy has been cultured in vitro as explants. The total accumulation in tissue and culture medium of the whey proteins alpha-lactalbumin and beta-lactoglobulin has been measured using specific radioimmunoassays. The control, uncultured tissue showed progressive morphological development from sparse, non-secretory epithelial tissue on day 60 to full lobulo-alveolar development with some accumulated secretion from day 100. In uncultured explants beta-lactoglobulin could be detected consistently from day 90 (13 +/- 12 ng/micrograms DNA, n = 4) and alpha-lactalbumin from day 100 (1.3 +/- 0.5 ng/micrograms DNA, n = 11). At all stages of pregnancy, both whey proteins increased markedly during the period of culture (up to 7 d). Stimulation of alpha-lactalbumin appeared to be primarily under prolactin control. Prolactin increased alpha-lactalbumin accumulation to a similar extent alone, or in the presence of insulin and/or corticosterone. The response to prolactin was dose-dependent over the range 0.4-20 nM (10-500 ng/ml). Porcine prolactin was more potent than ovine prolactin. There was no effect of porcine growth hormone and no synergism detected between prolactin and tri-iodothyronine. By contrast, no specific hormonal requirements were established for accumulation of beta-lactoglobulin, which appeared to increase in vitro if tissue remained viable in various combinations of insulin, corticosterone and prolactin. It was not stimulated by growth hormone. There was some indication of a prolactin-sensitive component in longer term cultures after day 4.

Effects of insulin, hydrocortisone and prolactin on cell morphology, growth and survival of mammary organoids from mid-pregnant mice cultured on collagen gels.

Mammary epithelial organoids consisting of groups of lobular-alveolar acini were prepared from mid-pregnant mice and cultured for 24, 48, 96 and 192 hr on attached collagen gels in the presence of combinations of insulin, hydrocortisone and prolactin. The organoids rapidly attached to the gels and with all the combinations of hormones used colonies of cells spread out as a monolayer from the organoids within 48 hr. Although colony formation continued for up to 192 hr in culture, the maintenance of parental organoid structure after 96 and 192 hr was strongly favoured when hydrocortisone was present in the culture medium. The presence of hydrocortisone produced a dose-dependent increase in the amount of organoid DNA associated with the collagen substratum but decreased the rate of DNA synthesis by the organoids, as measured by the incorporation of labelled thymidine into DNA, in a dose-dependent manner under these conditions. The results suggest that the presence of hydrocortisone minimised the loss of cells from the collagen matrix in these cultures.

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: correlation with tumour type and progression.

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.

Lactational transfer of 3,3',4,4'-tetrachloro- and 2,2',4,4',5,5'-hexachlorobiphenyl induces cytochrome P450IVA1 in neonates. Evidence for a potential synergistic mechanism.

On the first day of lactation, material rats were treated with a single low dose of 5 mg/kg body weight of 3,3',4,4'-tetrachlorobiphenyl (TCB) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) or with a combination of both congeners. Lactational transfer of these polychlorinated biphenyls (PCBs) was found in neonates and significant increases in microsomal cytochrome P450, cytochrome b5 and in glutathione-S-transferase activity were observed. Treatment with HCB did not increase neonatal ethoxyresorufin-O-de-ethylation (EROD) activities whereas a more than 26-fold increase in EROD activity was noted in response to exposure to TCB. However, EROD activities were increased more than 65-fold in response to the combined exposure to TCB and HCB. Exposure via milk to TCB caused a significant reduction in the N-demethylation of aminopyrine, but the combined exposure to TCB and HCB produced a significant reduction in the N-demethylation of dimethylnitrosamine. Lactational transfer of either TCB or HCB reduced marginally peroxisomal enzyme activities; however, exposure to a combination of TCB and HCB resulted in the highly significant reduction in KCN-insensitive palmitoyl-CoA oxidation and acetyl-CoA oxidation. Contrary to the reduction of these enzyme activities, the specific concentrations of CYP4A1 were significantly increased when neonates were exposed to either TCB or HCB. The largest induction, however, was observed in response to the combined exposure to both PCBs. Evidence is presented to suggest an induction of CYP4A1 which may be independent of the molecular substitution pattern of the two PCBs used in our studies but on a possible mode of synergistic interaction.

Distribution and elimination in vivo of polychlorinated biphenyl (PCB) isomers and congeners in the pigeon.

1. Pigeons were injected with a single dose of commercial PCB mixtures (Aroclor 1248 plus Aroclor 1260), killed 120 h later and the abundance of individual PCBs was determined in adipose tissue, gonads, liver, brain, kidney, heart, muscle and blood. 2. Elimination factors for individual PCBs were calculated. Values of greater than 1 were obtained for PCBs with meta-para-unsubstituted carbon atoms in at least one ring, indicating that elimination exceeded accumulation in all or most tissues. By contrast, ortho-meta unsubstituted PCBs had elimination factors less than 1, thus indicating their impaired removal. 3. Tissues with high microsomal monooxygenase activity had the highest elimination factors for individual PCBs (i.e. liver greater than kidney greater than muscle greater than heart). 4. Distribution of individual PCBs was independent of sex and of ortho-chlorine substitution and showed that 90% of total PCBs in cadavers was present in adipose tissue, 2% in kidneys, 1% each in brain, muscle and heart and less than 0.1% in blood. 5. The distribution of the highly toxic non-ortho and mono-ortho substituted PCBs did not differ amongst all tissues analysed. 6. The present studies indicate that elimination of PCBs in vivo is favoured by the molecular feature of unsubstituted meta-para carbon atoms in the biphenyl moiety.

Evidence for the induction of fatty acid desaturation in proliferating hepatic endoplasmic reticulum in response to treatment with polychlorinated biphenyls. Are fatty acid desaturases cytochrome P-450-dependent monooxygenases?

1. Polychlorinated biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem which accumulate in biological systems. 2. We have shown previously (Borlakoglu et al., 1990; Eur. J. Biochem. 118, 327-332) that 120 hr after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body weight, hepatic microsomal membranes showed significant increases in the proportion of arachidonate (20:4,5, 8,11,14), in the concentration of cytochrome P-450 and in the activities of a wide range of cytochrome P-450-dependent enzymes involved in the metabolism of drugs and other xenobiotics. 3. After treating pigeons and rats in vivo with Aroclor 1254, linoleate desaturases activity increased significantly 3.35-, 4.35-, 5.83- and 8.61-fold 24, 48, 68 and 120 hr for pigeons and 2- and 7-fold for rats respectively 48 and 120 hr post treatment. The total activity of linoleate desaturases in the whole liver of pigeons and rats increased 40- and 10-fold respectively. 4. There were excellent correlations between the concentrations of cytochrome b5 and cytochrome P-450 and the activity of pigeon linoleate desaturases. Extrapolation of the concentration of cytochrome P-450 to zero is coincident with zero linoleate desaturase activity. 5. Evidence is presented to suggest the novel concept that linoleate desaturation is dependent upon the catalytic cycle of these monooxygenases.

Induction of hepatic cytochrome P-450IA1 in pigeons treated in vivo with Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs).

1. Treatment with a commercial mixture of polychlorinated biphenyls (PCBs) resulted in highly significant increases in pigeon hepatic microsomal proteins (100-fold), cytochrome P-450 (11-fold), cytochrome b5 (7-fold), NADPH-cytochrome c-(P450) reductase (7-fold), ethoxycoumarin-O-deethylation (9-fold), aldrin epoxidase (22-fold), ethoxyresorufin-O-deethylation (48-fold), N-demethylation of dimethylnitrosamine (28-fold) but not of lauric acid 12-hydroxylation. 2. SDS-PAGE analysis of pigeon hepatic microsomal proteins induced by Aroclor 1254 suggested highly significant increases in the density of staining in bands of estimated Mr 51-52 kD, 54-54.5 kD, 57-58 kD, 59-60 kD and of 77.5-78.5 kD. 3. The induction of cytochrome P-450IA1 was confirmed by Western immunoblotting using the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4. 4. There was agreement between the 8-fold increase in cytochrome P-450IA1 increased staining of microsomal proteins, as judged by SDS-PAGE, and the 24-fold increase in the amount of protein that reacted with the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4, as judged by Western immunoblotting. 5. It is concluded that treatment with a commercial PCB mixture resulted in the induction of several isoforms of pigeon hepatic cytochrome P-450 in a fashion that is likely to be similar to that reported for mammals.

Metabolism of [14C]4-chlorobiphenyl by hepatic microsomes isolated from razorbills, pigeons and rats.

1. Analysis of individual PCB-isomers and congeners in extracts of adipose tissue from N = 12 razorbills suggested that 4-chlorobiphenyl was subjected to metabolism. 2. In vitro metabolism studies using [14C]-4-chlorobiphenyl as substrate showed that razorbills metabolise this substrate to [14C]4-chloro-4'-hydroxybiphenyl at an average rate of 20 pmol/mg microsomal protein/min. For comparison, the metabolism of [14C]-4-chlorobiphenyl by pigeons and rats was also studied, and average rates in the formation of [14C]-4-chloro-4'-hydroxybiphenyl of 12 pmol/mg microsomal protein min and 342 pmol/mg microsomal protein min were estimated. 3. A comparison of the hepatic drug metabolising enzyme system of razorbills and pigeons showed similar concentrations of cytochrome P-450, cytochrome b5 and comparable catalytic activities of cytochrome P-450-dependent monooxygenase, when assessed for HHDN epoxidase, PROD, EROD and the Phase II enzymes glutathiones-S-transferase, but were significantly lower, when compared with rats. The results obtained suggest fundamental differences in the catalytic activities of cytochrome P-450-dependent monooxygenase between avian and mammalian species. 4. The present study, however, provides evidence that fish-eating seabirds have the ability to metabolically dispose of certain PCB isomers and congeners, which are amongst the most ubiquitously distributed pollutants in the ecosystem.

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.

Effects of molecular substitution patterns on the cytochrome P-450-dependent metabolism of 2,2',3,5,5',6- and 2,2',3,4,4',6-hexachlorobiphenyl by rat liver microsomal monooxygenases.

The metabolism by rat hepatic microsomal cytochrome P-450-dependent monooxygenases of several model substrates that are specific for individual isoforms of cytochrome P-450 and the metabolism by these monooxygenases of two structurally related isomers of hexachlorobiphenyl was studied. The most striking result was that 2,2',3,5,5',6-hexachlorobiphenyl was metabolised in vitro at the rate of 4.5 pmol/mg microsomal protein per min, whereas the other isomer 2,2',3,4,4',6-hexachlorobiphenyl was not metabolised at detectable rates. This finding provides strong evidence for a regioselective oxidative attack by cytochrome P-450-dependent monooxygenase with preferential insertion of oxygen at meta-para unsubstituted carbon atoms. Investigations into the mechanism of this oxidative attack suggest that the ortho hydrogen atom at carbon atom C-6' of 2,2',3,4,4',6-hexachlorobiphenyl was associated with a lower charge (0.075 e) compared with the meta or para hydrogen atoms at carbon atom C-3' and C-4' of 2,2',3,5,5',6-hexachlorobiphenyl (0.086 e). In addition, measurement of the main C-C bond length using MOPAC calculations and X-ray crystalographic data suggests significant differences in the bond-length distance, with the main bond lengths of 1.390, 1.385 and 1.374 A, respectively, for bridgehead to ortho (C1-C2), for ortho to meta (C2-C3), and for meta to para bonds. These results provide evidence that the preferential meta-para oxidative attack is linked to a shorter carbon-carbon bond length and a more positive charge distribution of the corresponding hydrogen atoms.

Transport and cellular uptake of polychlorinated biphenyls (PCBs)--I. Association of individual PCB isomers and congeners with plasma lipoproteins and proteins in the pigeon.

Polychlorinated biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB-contaminated lipid components of food chains. PCBs are lipophilic and lipid-rich lipoproteins provide an excellent system to transport PCBs to tissues. We report here the distribution of PCBs between plasma fractions in the pigeon. Twenty-four hours after injection, [14C]4-monochlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl were associated with the protein-rich HDL fraction and the lipoprotein-poor fraction (predominantly albumin), rather than with the lipid-rich VLDL and LDL fractions. Five days after injection with the commercial PCB mixture Aroclor 1254, there was a distinctive distribution between the plasma fractions of the 41 congeners detected. Avian species have a poorly developed lymphatic system and dietary lipids are secreted into the portal vein. To emphasize this route of entry, the lipoprotein particles formed are termed portomicrons rather than chylomicrons. The most striking result was that the lipid-rich portomicron and the VLDL fraction was associated almost exclusively with only one congener (2,2',4,4'5,5'-hexachlorobiphenyl), whereas the other isomers and congeners were distributed amongst the LDL, HDL and the lipoprotein-poor (predominantly albumin) fractions. Thirteen of the congeners detected accounted for 74, 53 and 54%, respectively, of the total amount of PCBs in the LDL, HDL and lipoprotein-poor protein fractions. Five congeners that are highly toxic were enriched in the latter fraction. The distribution of PCBs is more complex than can be explained solely by their solubility in the lipid components of plasma fractions, and may suggest a complex association with apolipoproteins and plasma proteins that are important in transporting PCB to tissues. The identification of individual PCBs in lipoprotein fraction provides evidence for their role in the transport of lipophilic xenobiotics in blood and it is suggested that PCBs associated with lipoproteins are taken up by cells as lipoprotein-PCB complexes.

Transport and cellular uptake of polychlorinated biphenyls (PCBs)--II. Changes in vivo in plasma lipoproteins and proteins of pigeons in response to PCBs, and a proposed model for the transport and cellular uptake of PCBs.

The complex distribution of polychlorinated biphenyl (PCB) isomers and congeners amongst plasma fractions of the pigeon suggests that the lipid and apolipoprotein components of lipoproteins, as well as plasma proteins, may be important in transporting PCBs to tissues (Borlakoglu et al., Biochem. Pharmac. 40, 265 (1990]. Pigeons were injected with the commercial PCB mixture Aroclor 1254 (1.5 mmol/kg body weight). After 120 hr triacylglycerol-like droplets accumulated in hepatocytes ('fatty liver syndrome'), there was proliferation of the hepatic smooth endoplasmic reticulum, and plasma concentrations of triacylglycerol and total cholesterol increased. This was accompanied by significant decreases in plasma concentrations of total protein, total apolipoproteins of the low density lipoprotein (LDL) and the high density lipoprotein (HDL) fractions, and albumin and by a significant increase in that of urea, indicating increased protein breakdown. These results suggest that Aroclor 1254 increased hepatic lipid synthesis, but decreased hepatic production of albumin and apolipoproteins. This would explain the accumulation of triacylglycerol in the liver and the increase in the proportion of triacylglycerol to apolipoprotein in the total lipoproteins. From the evidence presented, a model is proposed based on the association of PCBs with hydrophobic domains of lipids and proteins for the transport of PCBs by plasma fractions, their uptake into cells and intracellular metabolism, and their accumulation in adipose tissue.