Brief Communication: Gas Contaminant Inhibition of Mammary Gland Differ-
entiation In Vitro 1,2 M. R. Warner and D. Medina
3.4
SUMMARY-A gas mixture of 95% 02/5% C02 from several commercial sources was contaminated with a filterable substance(s) that inhibited mammary gland morphologic differentiation in vitro.-J Natl Cancer Inst 54: 12571258, 1975.
THE MAMMARY DIFFERENTIATIVE PROCESS responds to a variety of influences in vitro (1); this reflects its usefulness as a sensitive assay system for the effects of toxic or carcinogenic compounds in vivo (2,3). . Recently, we were unable to repeat some of our earlier experiments on the induction of morphologic differentiation in vitro in mammary glands of hormonally pretreated mice (4). Since other laboratories had simultaneously reported success with this system (5), we persisted in our efforts and were finally rewarded. The limiting factors for our system may be relevant for other workers studying differentiation in vitro and may affect a variety of in vitro assay systems. MATERIALS AND METHODS
Three- to 4-week-old BALB/cCrgl mice were pretreated with 1 J.Lg 17~-estradiol and 1 mg progesterone in gum arabic suspension daily for 9 days (6). The day after the last injection, the mice were killed. Their skin was swabbed with 70% ethanol, a mid ventral incision was made, and the second and fourth mammary glands were dissected free from muscle and fascia under a dissection microscope (7). Glands were transferred to lens-paper rafts in Falcon plastic organ culture dishes (Bioq uest #3010) containing 1 ml Waymouth's medium MB752fl (8) with 350 J.Lg l-glutamine/ml; 35 J.Lg penicillin/ml; 1 J.Lg-aldosterone (Sigma #63C-0450)/ml; 5 J.Lg insulin/(Calbiochern #200972) /ml, and 5 J.Lg/ml ovine prolactin (NIH-P-S-8)/ml. Hormones were prepared as described by Rivera (1). Tissues were cultured for 5 days at 37° C in 95% O 2/5% CO 2 (Big 3, Ohio, or Matheson) in a water-jacketed incubator. Medium was changed the 3d day. Terminated cultures were fixed in Tellyesniczky's fluid and processed for whole mounts as described by Medina (3). Probability was computed by the Mann-Whitney U test according to Siegel (9). Glands were evaluated by the method of Meskimen (4) on a four point scale: O=no lobules; 1 = less than 50% of the mammary gland showed lobuloalveolar differentiation; 2 = 50% of the mammary gland showed lobuloalveolar differentiation; 3 = more than 50% of the mammary gland showed lobuloalveolar differentiation; 4 = 75% or more of the mammary gland showed lobuloalveolar differentiation. RESULTS
Twenty-four unsuccessful experiments were performed in 2 years. Differentiation was not obtained,
but maintenance of ducts and alveoli was routinely seen in all experiments. A number of variables were examined, including chemical and medium quality glassware contamination, water source, equipment contamination, and mouse strain. The experiments were unsuccessful in both a cosmopolitan environment at Houston and a rural environment at College Station, Bryan, Texas, in the laboratories of Dr. R. Cain. Subsequently, we obtained compressed 02/C02 from another area about 400 miles away (San Antonio), but were still unable to induce lobuloalveolar differentiation. Personal communication with a local representative of the Matheson Company indicated that the bottled gases used in both Houston and Bryan were probably produced in the same place. A charcoal filter was added to locally obtained gas with no noticeable increase in differentiation (Matheson Gas Products model 460 purifier and model 463 activated charcoal cartridge, for removal of oil and heavy hydrocarbons). Finally, with the addition of an HEPA filter to the charcoal filter [Mine Safety Co. # 15-85759 MSA ultrafilter airline filter housing assembly with #15-79030 HEPA filter (99.98% efficient for 0.3-J.L particulates), and with #15-46727 carbon filter (to remove light concentrations of organic vapors and acid gases)], we obtained normal morphologic levels of lobuloalveolar differentiation in three successive experiments (table 1). These experiments did not differ significantly in percent differen tia tion. DISCUSSION
Between 1962 and 1966, we performed 15 experiments in which good lobuloalveolar differentiation was obtained in mammary glands of BALB/cCrgl mice (4). A comparison of the morphology of our past experiments and those of Banerjee et al. (5), Ichinose and Nandi (6), and Singh and Bern (10) with morphology of the glands reported in the present series indicated that we obtained typical levels of morphologic differentiation using filtered gas. Biochemical similarity of tissues has not been assayed. A charcoal filter alone did not permit differentiation. No experiments were performed with only an HEPA filter. We attempted to obtain analyses of the commercial gas mixture to identify the toxic contaminant. Inquiries from our local gas suppliers (Big Three, Airco, Received December 26, 1974; accepted January 28, 1975. Partially supported by Public Health Service grants CA1l944 and CA10893 from the National Cancer Institute. 3 Cell Biology Department, Baylor College of Medicine, Houston, Tex. 77025. 4 We thank Drs. E. Rivera and B. K. Nair for their suggestions. We are also grateful to Dr. R. Cain, who generously allowed us to use his laboratory facilities. 1 2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VQL. 54, NO.5, MAY 1975
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WARNER AND MEDINA
TABLE I.-Extent of differentiation and repeatability of experiments Experiment No.
Percent of explants at differentiation grade
Number of explants
entiation In Vitro 1,2 M. R. Warner and D. Medina
3.4
SUMMARY-A gas mixture of 95% 02/5% C02 from several commercial sources was contaminated with a filterable substance(s) that inhibited mammary gland morphologic differentiation in vitro.-J Natl Cancer Inst 54: 12571258, 1975.
THE MAMMARY DIFFERENTIATIVE PROCESS responds to a variety of influences in vitro (1); this reflects its usefulness as a sensitive assay system for the effects of toxic or carcinogenic compounds in vivo (2,3). . Recently, we were unable to repeat some of our earlier experiments on the induction of morphologic differentiation in vitro in mammary glands of hormonally pretreated mice (4). Since other laboratories had simultaneously reported success with this system (5), we persisted in our efforts and were finally rewarded. The limiting factors for our system may be relevant for other workers studying differentiation in vitro and may affect a variety of in vitro assay systems. MATERIALS AND METHODS
Three- to 4-week-old BALB/cCrgl mice were pretreated with 1 J.Lg 17~-estradiol and 1 mg progesterone in gum arabic suspension daily for 9 days (6). The day after the last injection, the mice were killed. Their skin was swabbed with 70% ethanol, a mid ventral incision was made, and the second and fourth mammary glands were dissected free from muscle and fascia under a dissection microscope (7). Glands were transferred to lens-paper rafts in Falcon plastic organ culture dishes (Bioq uest #3010) containing 1 ml Waymouth's medium MB752fl (8) with 350 J.Lg l-glutamine/ml; 35 J.Lg penicillin/ml; 1 J.Lg-aldosterone (Sigma #63C-0450)/ml; 5 J.Lg insulin/(Calbiochern #200972) /ml, and 5 J.Lg/ml ovine prolactin (NIH-P-S-8)/ml. Hormones were prepared as described by Rivera (1). Tissues were cultured for 5 days at 37° C in 95% O 2/5% CO 2 (Big 3, Ohio, or Matheson) in a water-jacketed incubator. Medium was changed the 3d day. Terminated cultures were fixed in Tellyesniczky's fluid and processed for whole mounts as described by Medina (3). Probability was computed by the Mann-Whitney U test according to Siegel (9). Glands were evaluated by the method of Meskimen (4) on a four point scale: O=no lobules; 1 = less than 50% of the mammary gland showed lobuloalveolar differentiation; 2 = 50% of the mammary gland showed lobuloalveolar differentiation; 3 = more than 50% of the mammary gland showed lobuloalveolar differentiation; 4 = 75% or more of the mammary gland showed lobuloalveolar differentiation. RESULTS
Twenty-four unsuccessful experiments were performed in 2 years. Differentiation was not obtained,
but maintenance of ducts and alveoli was routinely seen in all experiments. A number of variables were examined, including chemical and medium quality glassware contamination, water source, equipment contamination, and mouse strain. The experiments were unsuccessful in both a cosmopolitan environment at Houston and a rural environment at College Station, Bryan, Texas, in the laboratories of Dr. R. Cain. Subsequently, we obtained compressed 02/C02 from another area about 400 miles away (San Antonio), but were still unable to induce lobuloalveolar differentiation. Personal communication with a local representative of the Matheson Company indicated that the bottled gases used in both Houston and Bryan were probably produced in the same place. A charcoal filter was added to locally obtained gas with no noticeable increase in differentiation (Matheson Gas Products model 460 purifier and model 463 activated charcoal cartridge, for removal of oil and heavy hydrocarbons). Finally, with the addition of an HEPA filter to the charcoal filter [Mine Safety Co. # 15-85759 MSA ultrafilter airline filter housing assembly with #15-79030 HEPA filter (99.98% efficient for 0.3-J.L particulates), and with #15-46727 carbon filter (to remove light concentrations of organic vapors and acid gases)], we obtained normal morphologic levels of lobuloalveolar differentiation in three successive experiments (table 1). These experiments did not differ significantly in percent differen tia tion. DISCUSSION
Between 1962 and 1966, we performed 15 experiments in which good lobuloalveolar differentiation was obtained in mammary glands of BALB/cCrgl mice (4). A comparison of the morphology of our past experiments and those of Banerjee et al. (5), Ichinose and Nandi (6), and Singh and Bern (10) with morphology of the glands reported in the present series indicated that we obtained typical levels of morphologic differentiation using filtered gas. Biochemical similarity of tissues has not been assayed. A charcoal filter alone did not permit differentiation. No experiments were performed with only an HEPA filter. We attempted to obtain analyses of the commercial gas mixture to identify the toxic contaminant. Inquiries from our local gas suppliers (Big Three, Airco, Received December 26, 1974; accepted January 28, 1975. Partially supported by Public Health Service grants CA1l944 and CA10893 from the National Cancer Institute. 3 Cell Biology Department, Baylor College of Medicine, Houston, Tex. 77025. 4 We thank Drs. E. Rivera and B. K. Nair for their suggestions. We are also grateful to Dr. R. Cain, who generously allowed us to use his laboratory facilities. 1 2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VQL. 54, NO.5, MAY 1975
1257
1258
WARNER AND MEDINA
TABLE I.-Extent of differentiation and repeatability of experiments Experiment No.
Percent of explants at differentiation grade
Number of explants
