Introduction

Abstract Introduction References

There is a small but intriguing body of literature that suggests an association between opioids and fetal lung maturation. Retrospective clinical studies suggest that infants born to narcotic-addicted mothers have a decreased risk of respiratory distress syndrome (RDS) (1). In addition, animal studies have shown that administration of heroin or morphine to pregnant animals or their fetuses results in accelerated fetal lung maturation (4). Given the finding of opioid receptors and endogenous opioids (endorphins) in humans and of specific opioid receptors in lung tissue of several species (8, 9), these data suggest that endogenous opioids may play a role in the process of fetal lung development. Nevertheless, virtually all of these studies have used in vivo systems or are based on retrospective studies of human pregnancies, leaving open the question of whether the observed effects are the result of a direct effect of the opioids studies or, rather, a secondary phenomenon derived from the influence of opioids on other hormones, such as glucocorticoids, thyroid hormone, or prolactin (10), known to be important modulators of fetal lung maturation (15).

An association between cocaine abuse and reduction in RDS has also been reported (16, 17), although this has not been a consistent finding (18, 19). In vivo animal studies have also suggested that maternal cocaine administration accelerates fetal lung maturation in rats and rabbits (20, 21), although these results may also be related to the effect of cocaine on glucocorticoid or catecholamine metabolism (21, 22).

The present study was undertaken to resolve these questions by studying the direct effect of opioids and cocaine on fetal rat lung explants and isolated type II cells in culture.

	Materials and Methods

Fetal Lung Explant Preparation and Culture Conditions

Timed-pregnant Sprague-Dawley rats (Charles River, Wilmington, MA) were killed on Days 18, 19, or 20 of gestation (day of mating = time 0), and fetal lungs were removed under sterile conditions and dissected free of nonpulmonary tissue. Whole lungs were then chopped into 0.7-mm³ cubes and cultured for 44 h in a continuously rocking chamber with an atmosphere of 95% O₂:5% CO₂ at 37°C, after the method of Gross and colleagues (23). In experiments involving morphologic analysis, right upper lung lobes were identified and prepared for explant culture as described previously. Explants were placed into tissue culture dishes containing F-12 media (Gibco Labs, Grand Island, NY) to which were added heroin, morphine, methadone, naloxone, or cocaine in a series of doses ranging from 1 × 10⁸ M to 1 × 10³ M.

Type II Cell Isolation and Culture Conditions

Timed-pregnant Sprague-Dawley rats were killed on Day 20 of gestation and fetal lungs were removed en bloc under sterile conditions. The fetal lungs were minced and washed in Hanks' medium (Gibco), placed in a solution containing 0.25% trypsin (Worthington, Freehold, NJ) and 2 mg/ml DNase I (Sigma, St. Louis, MO), and dissociated in a 37°C water bath using a Teflon stirring bar. The cell suspension was then passed through a 40-µm Nitex filter into chilled minimum essential medium (MEM; Gibco) containing 20% fetal bovine serum (FBS). Three successive differential adherence steps (24) were used to separate type II cells from fetal lung fibroblasts. These methods yielded type II cell cultures containing ~ 85-90% epithelial cells (as determined using cytokeratin staining), as previously described (25); viability of the epithelial cells in culture was 88 to 95%. The isolated type II cells were again centrifuged and the pellet was kept in a 37°C water bath for 1 h, then resuspended, plated at a final concentration of ~ 2.5 × 10⁶/ml, and cultured for 44 h at 37°C in a 5% CO₂ incubator (24) in MEM with 2% FBS to which was added varying amounts (1 × 10⁸ M to 1 × 10³ M) of heroin, morphine, methadone, or cocaine.

Incorporation Studies

After 44 h, culture medium was removed from the dishes containing the explants or type II cells and was replaced with medium containing 2 µCi/ml [methyl-³H]choline (New England Nuclear, Wilmington, DE) without opioids or cocaine. After a 4-h pulse period, the explants or type II cells were washed free of radiolabeled [³H]choline in ice-cold 0.9% saline and sonicated. Lung lipids were extracted according to a modification (26) of the method of Bligh and Dyer (27) and phosphatidylcholine (PC) was isolated using thin-layer chromatography (TLC) on Baker Si-250 silica gel plates (28). Disaturated PC (DSPC) was also isolated by TLC (28) after oxidation with osmium tetroxide following the method of Mason and associates (29). After separation of the PC and DSPC and visualization by exposure to iodine vapor, the radiolabeled phospholipid-containing spots were scraped into scintillation vials for determination of the rate of [³H]choline incorporation into PC and DSPC. Results are expressed as picomoles per hour per milligram of protein. Protein was assayed using the method of Lowry and coworkers (30) or the micromethod of Bradford (31) for the explants and type II cells, respectively.

Viability and Toxicity Studies

Type II cell viability was determined using trypan blue exclusion. Lactate dehydrogenase (LDH) release into the medium over the 2-d explant culture period was determined on an Ektachem 700 Analyzer (Eastman Kodak, Rochester, NY) using the pyruvate-to-lactate reaction (32).

Morphologic Analysis

Right upper lobe lung explants, 19 d old, were cultured for 44 h in culture with and without opioids, as described previously. The explants were then fixed overnight in 2.3% glutaraldehyde and 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in calcium chloride buffer for 1 h, and dehydrated through a graded series of alcohols (33); tissue blocks were then embedded in Epon and thin sections were stained with uranyl-lead hydroxide. Electron microscopy was then performed, and random sections were photographed at ×3,000 magnification. Type II cells were identified by the presence of lamellar bodies (LB), counted, and expressed as the ratio of type II cells or LB per alveolar lining cell (ALC) (34).

This protocol was approved by the Institutional Animal Care and Utilization Committee of the University of Maryland School of Medicine. Student's t test, the Mann-Whitney rank-sum test, with Bonferroni corrections, or analysis of variance with Dunnett's post hoc test, when appropriate, were used to determine statistical significance. Results are expressed as means ± SEM.

	Results

Morphine and heroin, at a dose of 1 × 10³ M, and methadone at a dose of 1 × 10⁴ M, resulted in a significantly increased incorporation of choline into PC and DSPC by the fetal lung explants derived from both 19- and 20-d gestations (Figures 1a and 1b), with effects being slightly greater (increases ranging from 75 to 120% for the three drugs tested) in the 19-d explants than in the 20-d explants (55 to 80% increases). When doses one-half order of magnitude lower (5 × 10⁴ M for heroin and morphine and 5 × 10⁵ M for methadone) were used, smaller (35 to 60%) increases were noted compared with control values; no stimulatory effects were seen with all doses  1 × 10⁴ M for heroin and morphine and  1 × 10⁵ M for methadone (data not shown). Preincubation with equimolar doses of naloxone did not block the stimulatory effect of the opiates, with naloxone plus methadone, heroin, and morphine yielding values compared with opiates alone of 92 ± 4%, 102 ± 7%, and 86 ± 4%, respectively (P = NS). No significant opioid effect was seen in Day-18-derived explants. Cocaine, at doses ranging from 10⁸ to 10³ M, had no effect on the rate of choline incorporation into PC or DSPC on any day studied.

View larger version (40K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   Figure 1.   Effects of opioids and cocaine on the rate of choline incorporation into PC (a) and DSPC (b) by lung explant tissue derived from Day 19 and 20 fetal rat lung. +P < 0.005; *P < 0.001 versus same-day control; n = 4-12.

Figure 2 shows representative photomicrographs from control and heroin-, methadone-, and morphine-treated explants. Approximately 3-fold increases in type II cells per alveolar lining cell and 5-6-fold increases in LB per ALC were present in each of the opioid-treated groups. In addition, there were more LB per type II cell in the opioid-treated explants (Table 1).

View larger version (157K): [in this window] [in a new window]   View larger version (153K): [in this window] [in a new window]   View larger version (157K): [in this window] [in a new window]   View larger version (158K): [in this window] [in a new window]   Figure 2.   Representative photomicrographs of explants derived from right upper lobes of 19-d fetal rats in culture for 44 h. (a) Control; (b) heroin-treated (103 M); (c) morphine-treated (103 M); (d) methadone-treated (104 M). Final magnification, ×3,000; bar = 5 µm. Note the paucity of LB in control versus opioid-treated explants.

Isolated type II cells also responded to treatment with opioids, with an ~ 50% increase in choline incorporation into PC; P < 0.01 (Figure 3).

View larger version (36K): [in this window] [in a new window]   Figure 3.   Effect of opioids on choline incorporation into PC by isolated type II cells derived from Day-20 fetal rat lung. Results are expressed as percentage of control. *P < 0.01 versus control; n = 6.

There were no significant differences between control and treated groups in type II cell viability, as determined by trypan blue exclusion. Control cells excluded dye in 91.8 ± 2.7% of the cases versus 86.1 ± 2.1% for heroin-treated, 90.2 ± 3.6% for morphine-treated, and 86.4 ± 3.8% for methadone-treated cells (P = NS). There were also no significant differences in LDH release into the medium by opioid-exposed explants.

	Discussion

It is generally accepted that infants born to opiate-addicted mothers have a lower incidence of RDS, although supporting data are sparse (1, 2). In a retrospective study of the prevalence of RDS in infants of heroin-addicted and nonaddicted mothers, Glass and colleagues (2) noted that no RDS developed in 33 consecutive premature (32- to 37-wk gestation) infants born to addicted mothers, as opposed to 26 cases of RDS in 123 consecutive nonaddicted pregnancies. This clinical impression was supported by the finding of increased lecithin/sphingomyelin ratios in the amniotic fluid of mothers with a history of narcotic addiction in some (3) but not all (35) studies. Another retrospective study found no significant difference in the incidence of RDS between infants born to narcotic-addicted mothers and a control group (36).

In an effort to clarify the relationship between narcotics and lung maturation, in vivo animal models were used (4- 7). With heroin (~ 1-3 mg/kg twice a day intravenously), Taeusch and associates (4, 5) found that after direct fetal (but not maternal) injection, the lungs were approximately 70% more distensible and retained 30 to 100% more air on deflation to low transthoracic pressures. Comer and associates (7) injected pregnant rabbits either with morphine sulfate (1 mg/kg/d for 10 d) or with naloxone (0.4-5 mg/d) and performed static pressure-volume measurements on fetal lungs at Day 28 of gestation (term = 31 d). Morphine treatment resulted in improvement of pulmonary mechanics and an increased air/tissue ratio by histologic analysis; naloxone injection had an inhibitory effect.

In a small prospective study of infants born at less than 34 wk of gestation, Zuckerman and coworkers (16) reported a decreased incidence of RDS in infants born to cocaine-addicted mothers (1 of 8 versus 13 of 25 control subjects). Other groups have not been able to confirm these findings (18, 19). In vivo animal studies suggest that maternal cocaine treatment can result in increased surfactant production and morphologic changes consistent with accelerated lung maturation (20, 21); however, the possibility that these effects are mediated through changes in circulating maternal and fetal glucocorticoid or catecholamine levels cannot be ruled out. Sosenko (21) noted significant increases in maternal and fetal corticosterone and total catecholamine concentrations in cocaine-treated rats.

The clinical studies cited above are limited in scope, largely uncontrolled and retrospective, and difficult to interpret because of the many variables involved in a drug-using population that are known to influence fetal lung development, including chronic stress, tobacco and alcohol use, malnutrition, maternal infection, fetal growth retardation, and race (15), and, of course, polydrug abuse. Moreover, both endogenous and exogenous opioids and cocaine modulate certain hormonal influences that are well known to affect fetal lung development, making it unclear whether an acceleratory effect in an in vivo model connotes a direct or indirect effect of opioids/cocaine. In sheep, leu-enkephalin administration results in increased fetal glucocorticoid levels (10); and in rats, cocaine has been shown to increase corticosterone by stimulation of the pituitary-adrenal axis with resultant adrenocorticotropin hormone (ACTH) release (22). Both endorphins (11) and morphine (37) result in the release of prolactin, which has also been implicated in fetal lung maturation (15). In addition, -endorphin and ACTH (both of which are derived from a precursor pro-opiocortin [38]) are secreted in parallel by the pituitary in response to stress and other ACTH-releasing stimuli. Thus, a close correlation between cortisol and -endorphin levels exists, further complicating the interpretation of in vivo human and animal studies.

Only a single previous study has investigated the effect of an opioid on fetal lung development in vitro, although that was not the primary focus of the steady. Smith and Torday (39) studied the effect of 10⁸ M heroin on monolayer cultures prepared from 28-d fetal rabbit lungs. The heroin-treated group incorporated 74% more choline into PC than did the control group; this increase did not reach statistical significance, possibly because of small sample size and considerable scatter. Furthermore, results using only a single, low concentration of heroin are given.

For these reasons we chose an in vitro approach for our experiments. In both explant culture and isolated type II cell culture, opioids accelerated fetal lung maturation. However, in both cases, supra-physiologic doses of opioids were needed to demonstrate the acceleratory effect. For example, for methadone the effective doses (5 × 10⁵ M to 1 × 10⁴ M) used in our study are about two orders of magnitude higher than serum methadone levels in patients on methadone maintenance regimens (40, 41), and for heroin and morphine (5 × 10⁴ M to 1 × 10³ M) about two to three orders of magnitude higher than serum levels in patients treated for chronic pain (42, 43) (although addicts using higher doses of heroin intravenously can attain higher peak serum levels). Nevertheless, our results do not appear to be nonspecific cytotoxic effects because neither LDH release by the explants nor trypan blue exclusion by the type II cells is adversely affected. Given the pharmacologic doses necessary to obtain the acceleratory effect and the inability to block these effects with naloxone, the effects noted in this study do not appear to be mediated via endogenous endorphin receptors. Studying the effect on lung development of endorphins specific to the various receptor classes and of specific receptor blocking agents may clarify this issue.

Our data suggest that the clinical impression of a lower incidence of RDS in infants born to heroin-addicted mothers reflects a direct effect on lung maturation rather than a secondary phenomenon derived from the influence of opioids on other hormones; the lack of an in vitro response to cocaine suggests that any effect of cocaine in previous animal studies and clinical reports is probably a secondary effect.