Raymond R. Dils

Isolation of the epithelial subcomponents of the mouse mammary gland for tissue-level culture studies

We have developed a tissue dissociation procedure for the mouse mammary gland whereby it is possible to isolate the parenchyma as intact structural subcomponents essentially free of mesenchyme. Whole mammary fat pads are coarsely minced and subjected to a limited collagenase digestion, with mechanical dissociation for 90 min at 37° C. The parenchyma is released as a mixture of multicellular organoids and monodispersed cells. Use of a graded series of filters (400, 250, 150, 95, and 51 μ pore size) allows the separation of parenchyma from nonparenchymal material, with a further enrichment of the former into ductal, ductal-lobular, and terminal end-bud or alveolar populations. Yield is variable and dependent upon the nature of the starting tissue, e.g., mouse strain, age, and parity. These organoid fractions may be established separately in culture. This procedure allows for the study of the ductal and terminal regions of the mouse mammary gland in culture at the tissue level.

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.

[61] Preparation and culture of mammary gland explants

Publisher Summary This chapter describes the preparation and culture of mammary gland explants. The mammary gland is an excellent tissue for studies on the hormonal control of growth and differentiation. The removal of endocrine glands in vivo followed by replacement therapy has given valuable insights into the considerable variation that occurs between species in their minimal hormonal requirements for the different phases of the processes. Histological studies showed that prolactin added to insulin and cortisol initiates or maintains secretory activity in vitro in mammary tissues from pregnant mice, TM and this key observation has subsequently been extensively exploited in biochemical studies. Lobuloalveolar mammary tissue can be obtained from midpregnant rabbits or from rabbits made pseudopregnant either by mating with a vasectomized male or by a single injection into the marginal ear vein of 50–100 IU of human chorionic gonadotropin in 1 ml of water or saline. It is found that mammary gland from pseudopregnant or from midpregnant rabbits shows good preservation of histological structure in media that contain insulin, or insulin and corticosterone, with an atmosphere of 95% O 2 –5% CO 2 .

Evidence for the induction of cytochrome P‐452 in rat liver by Aroclor 1254, a commercial mixture of polychlorinated biphenyls

We have examined the ability of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) to induce hepatic cytochrome P‐452‐linked enzyme activities in rat and pigeon liver five days after its intraperitoneal injection. The results provide evidence that, at the doses used, Aroclor 1254 induces cytochrome P‐452‐linked enzyme activities in rats, but not in pigeons. This inductive effect was previously regarded as being specific for hypolipidemic drugs and phthalate ester plasticisers.

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.

Hormonal induction of α-lactalbumin and β-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.

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.

Metabolism by rat hepatic microsomes of individual isomers and congeners in Aroclor 1016

Polychlorinate biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem. Commercial mixtures of PCBs (e.g. Aroclors) can contain up to 80 different isomers and congeners, many of which accumulate in the fatty tissues of species at the apex of food chains by the ingestion of PCB-contaminated lipid components of foods. Studies in vivo on the accumulation of individual isomers and congeners of commercial mixtures of PCBs in the adipose and other tissues of fish-eating seabirds, pigeons and rats have shown that individual PCBs that lack adjacent HG atoms in at least one of the rings are enriched in tissues, indicating that their accumulation exceeds their elimination by metabolism and excretion. The authors report here experiments designed to test whether these structural rules governing the accumulation or metabolism of individual PCBs in vivo are supported by measurements in vitro of the metabolism of individual PCBs in a commercial mixtures of PCBs by preparations of rat hepatic microsomes.