Toxicology, 71 (1992)257-266 Elsevier Scientific Publishers Ireland Ltd.

257

Placental aryl hydrocarbon hydroxylase activity and placental calcifications Guy Huel ", Franqoise Girard ~, Catherine Nessmann b, Jean Philippe Blot c, G6rard Br6art" and Thierry Moreau"

Godin '',

aEpidemiological Research Unit and Robert Debrk Hospital, bLaboratoire de Biologie du Dbveloppement et de la Reproduction and "Service d'Obst~trique, French National Institute of Health and Medical Research (INSERM) (France) (Received July 30th, 1991; accepted December 4th, 1991)

Summary Induction of aryl hydrocarbon hydroxylase (AHH) activity in the placenta as a result of maternal exposure to polycyclic aromatic hydrocarbons contained in cigarette smoke has been well documented. Furthermore, calcifications are more prevalent in the placentas of pregnant smokers than in those of non-smokers. The present study examines whether this latter relationship could be explained by the induction of AHH activity in the placenta. AHH levels were determined at birth in 141 unselected pregnant women admitted for delivery. Macroscopic placental examination was performed for vascular lesions, abnormalities of placental shape, of the cord and parameters of placental maturity such as basal and parenchymatous calcifications. Significant increases in the prevalence of calcifications of the placental basal plates and parenchyma with the induction of placental AHH were found. A similar significant association between smoking and AHH activation was also observed. These findings remained unchanged when controlling for smoking status assessed both by questionnaire and presence of cotinine in mother's urine. Moreover, the apparent association between smoking 'factor' and calcifications disappeared when controlling for AHH induction. Therefore, the association between smoking and placental calcifications previously related could be mediated by the AHH induction.

Key words." Aryl hydrocarbon hydroxylase; Human placenta: Placental calcifications

Introduction Induction of aryl hydrocarbon hydroxylase (AHH) activity in the placenta as a result of maternal smoking has been well documented [1-8]. This enzyme oxidizes various polycyclic aromatic hydrocarbons abundantly present in cigarette smoke. AHH activity may also be induced by a variety of polycyclic aromatic hydrocarbons, including benzo[a]pyrene, which are ubiquitous environmental pollutants produced by fuel combustion and factory emissions [9]. In addition to the aforementioned factors, It has been shown that passively inhaled tobacco smoke may induce placental Correspondence to: Guy Huel, Unit6 de recherches en 6pid6miologie, 16, avenue Paul Vaillant-Couturier, 94807 Villejuif Cedex, France. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

258

A H H activity [8]. A H H inducibility is associated with the harmful effects of environmental chemicals [10]. A large prospective study has previously demonstrated that subchorionic thrombosis and placental calcifications are more prevalent in the placentas of pregnant smokers than in those of non-smokers [11]. Furthermore, placental calcifications are often observed in normal pregnancies and may be associated merely with the normal maturation of the placenta [12]. This study of placental A H H metabolism was undertaken to examine whether there may be a relationship between A H H activity and some macroscopic placental characteristics. Methods

Sld~]ccls The study population consisted of unselected pregnant women under obstetrical care at the Baudelocque maternity clinic+ an obstetrical service in Paris, France. Information regarding tobacco exposure during and before pregnancy as well as family+ social and obstetrical histories were obtained from all study participants and recorded within the framework of the perinatal study of Baudelocque. At birth, placentas were collected for macroscopic examinations and assays of enzyme activity. Tobacco exposure was determined by direct postpartum interview the third day following delivery. Mothers were classified as smoker if they smoked more than one cigarette daily during the 3 trimesters of pregnancy. Women were considered as nonsmokers (controls) if they did not smoke during the last trimester .just before the beginning of pregnancy, if they did not smoke during one of the trimesters of pregnancy or if they were not passive smokers. More information on the design of the present study have been described elsewhere [8]. Mothers" urine was collected before the third day following delivery for nicotine and cotinine analysis.

Specimen storage Ji~r assa)' 0[ enzyme activity At birth, placental samples (2 x 10 g) were collected, placed in a polystyrene box, stored at -70°C and sent within 3 days to the laboratory for analyses of A H H activity [8]. Assay ~[ enzyme activit)" After thawing in 0.1 M potassium phosphate buffer (pH 7.4) and washing two times in the buffer, placental samples were homogenized with a glass-teflon homogenizer. The microsomes were then isolated by differential centrifugation. Homogenates were centrifuged at 27 000 x g for 20 min and the supernatants obtained were further submitted to centrifugation at 105 000 x g for 60 min. All these processes were performed in a cold room at 4°C. A H H activity was measured by the Nebert and Gelboin fluorimetric procedure [13]. The fluorometer was calibrated using quinine sulfate before each run, and the fluorescence was quantified in duplicate with 3-hydroxy-benzo[a]pyrene. One unit of A H H activity was defined as the amount of enzyme catalyzing the formation of a hydroxylated product during 30 min with a fluorescence equivalent to I pmol of 3-hydroxy-benzo[al)pyrene (3-OH-BP) [7]. As a result, the values were reported as pmol of 3-OH-BP formed

259 within 30 min (pmol 3-OH-BP/mg protein per 30 min). Information on the cotinine analysis in urine used in the present study has been described elsewhere [14].

Macroscopic placental examination At birth, the placentas were macroscopically examined alter formalin fixation. Placentas were weighed without membranes and cord and cut into parallel slices at l-cm intervals. Different aspects of placental pathology were taken into account: ( 1) vascular lesions (more than 3 infarcts, massive or basal perivillous fibrin deposition, retroplacental hematoma), (2) major abnormalities of placental shape (extrachorialis, multipartita), of the cord (velamentous insertion, single umbilical artery) and parameters of maturity (basal plate and parenchyma calcifications, subchorial thrombosis, subchorial or marginal perivillous fibrin deposition, marginal infarct, lobation, congestion and septa) following a protocol previously described [15]. Placental measurements were also recorded (weight, largest and smallest diameter, thickness). Statistical methods In agreement with a previous report [16] the distribution of A H H values was skewed and bimodal (Fig 1). Consequently classical parametric tests could not be applied. Quantitative non-parametric tests were also ruled out due to the large quantity of tied data. As a result we tested for trend with A H H activity. For this purpose we divided our sample into seven ordered levels of A H H (0-0.9, 1-1.9, 2-2.9, 3-4.9, 5-8.9, 9-49.9, 50 and over in pmol/mg protein per 30 min). The lower limit of each of these categories was chosen as the score for trend testing [17] concerning the existence of placental calcifications with increasing levels of AHH. If x k is the level of A H H activity in the class k, an appropriate statistic t\)r testing trend is to consider the regression of the deviations ( u k - ek) on .vk, where ak and e k are the observed

{30 ~i l i b

60

40 20

A.H.H. ( log scale) 0

0

~ 0 0 ~I I I i - ~ - - - N0 ~

~ ~

Q O ~~ Ol Oi ~l Ol ~l

~ ~

0

~

~ ~

Fig. 1. Frequency distribution (log scalc) of placental aryl hydrocarbon h>.droxylascactivit>, tin pmol 3-OH-BP/mg protein per 30 rain).

26~ and expected number of placenta respectively with or without calcifications by categ o r y , N t h e t o t a l n u m b e r a n d nl, no a n d mk t h e m a r g i n a l t o t a l s :

N3

I/:

Xk(a k -- ek)

/2

k=l ntn o

N

xfm k -

k=l

xkm k

k=l

w h i c h s h o u l d b e r e f e r r e d t o t a b l e s o f c h i - s q u a r e o n o n e d e g r e e o f f r e e d o m [18]. T h e c o m b i n e d effects w e r e e s t i m a t e d u s i n g t h e s a m e b a s i c t e c h n i q u e s w i t h a d j u s t m e n t b y s t r a t i f i c a t i o n , i.e. t h e r e l a t i v e risk f o r o n e f a c t o r b e i n g a d j u s t e d f o r t h e effects o f t h e o t h e r [17]. F o r t h e p u r p o s e s o f i l l u s t r a t i o n a n d to d e t e c t a p o t e n t i a l t h r e s h o l d effect, t h e level o f A H H e q u a l t o o r less t h a n o n e u n i t ( m o l e / m g p r o t e i n p e r 30 r a i n ) w a s c h o s e n as a b a s e l i n e . In t h i s m a n n e r r e l a t i v e risk w a s c a l c u l a t e d f o r e a c h c u m u l a t i v e level. T h e c u m u l a t i v e d i s t r i b u t i o n o f A H H is p r e s e n t e d in s u b s e q u e n t t a b l e s .

Results No significant relationship or association was found with AHH activity and vascular lesions, major abnormalities of the placenta, or shape of the cord. Length of gestation was not significantly related to AHH activity or placental calcifications.

TABLE I CUMULATIVE DISTRIBUTION LEVEL OF ARYL HYDROCARBON HYDROXYLASE ACTIVITY (pmol/mg protein per 30 min) IN PLACENTA ACCORDING TO THE EXISTENCE OF PLACENTAL CALCIFICATIONS OF THE MATERNAL SURFACE OR OF THE PARENCHYMA AHH levels Basal plate calcifications Relative risks"

No (,7) Yes (n)

0-1

_>1

->2

_>3

>_5

_>9

>_50

Total ~

53 31

32 25 1.33

25 20 1.36

19 19 1.71

8 16 3.41

4 10 4.27

2 5 4.27

85 56

12 12 3.20

7 7 3.20

4 3 2.40

105 36

X21df = 4.4 b" P < 0.05

Parenchyma calcifications Relative risks"

No (n) Yes (n)

64 20

41 16 1.24

30 15 1.60

24 14 1.86

X21d.f. = 2.4b: NS

~'Relative risks for each cumulative level of AHH distribution as the empirical odds-ratio for the corresponding 2 × 2 table. [17]. Level < I was choosen as baseline. bTest for trend [17]. CThis table represents the cumulative distribution level of AHH. Consequently the class >- I units included subjects of the class >- 2 which included subjects in class _>3 and so on.

261 Regarding the parameters of maturity, a positive trend was observed in the placentas, primarily with calcifications of the basal plate and/or of the parenchyma with A H H activity. No trend was found for the other parameters of maturity studied (subchorial thrombosis, subchorial, or marginal perivillous fibrin deposition, marginal infarct, lobation, congestion and septa). Table I shows the cumulative frequency distribution of A H H activity according to the existence of calcifications of the basal plate or the parenchyma. These cumulative frequency distributions were quite similar under 5 units (pmol/mg protein per 30 min). Above 5 units the relative risks increased as compared to the "non-calcifications" groups. These considerations were supported by the relative risk of calcifications with increasing A HH activity as shown in Fig. 2. A test for a trend between A H H activity and the frequency of placentas with basal plate calcifications was carried out, a significant trend was found (test for trend: Xeidr = 4.4, P < 0.05). This translates as an increase in the risk of calcification of the basal plate with increasing A H H activity. A similar pattern was observed for the parenchymal calcifications. Nevertheless, for the latter case, the results proved insignificant (test for trend: X2idr = 2.4: P > 0.05). The relative risk of observing placental activity increased sharply between 3 and 5 units for calcifications of both the parenchyma and basal plate. Values below 5 units were considered very low activity and close to the sensitivity limit of the method. Consequently in the following statistical analysis we refer to "induced placenta' if A H H activity was greater than or equal to 5 units and "non-induced placenta' if A H H activity was less than 5 units. The latter corresponds with the threshold activity of both the potential inducer (smoking) and the potential effect (calcifications). Dividing A H H into two classes (induced placenta or non-induced placenta), i.e. _>5 units and < 5 units respectively, demonstrated (Table II) a statistically signifi-

s!

Relltive

3.

~

Risks

2.

I

l • I

i •2

, •3

l )5

-l~

Basal calcif.

4-

P a r e n c h y . cal.

i )9

• .A.H.H.

Ictivity

pmole 3-OH-BP.mg pcoLein /30rnin.

Fig.2. Relativerisksofexceedingdifrerentvaluesofarylhydrocarbonh).drox',lascacti',ityaccordingto placentalcalcifications.

262 TABLE I1 DISTRIBUTION OF I N D U C E D PLACENTA A C C O R D I N G TO THE EXISTENCE OF PLACENTAL CALCIFICATIONS (BASAL PLATE AND PARENCHYMAt. Calcifications

Basal plate

Parenchyma

No

Yes

No

Induced placenta No (117) Yes 124t

77 8

40 16

93 12

24 12

Total

85

56

105

36

11411

Relative risks

2.6 (1.5-3.3) a X21df = 8.7

Yes

P < ().01

2.4 ( 1.3-4. I Ia X21dl = 9.1 P < I}.0l

After control for cotinine detection

X'ld i = 9.1 P < 0.0l

X21d i = 7.1 P < (l,01

After control for smoking status

X21d~ =

x~kt~ = 5.6 /' < il.02

5.5 P < i1.02

a95% confidence bounds.

cant

association

between

placental

0.01) a n d p l a c e n t a l p a r e n c h y m a

basal

plate calcifications

(X21dl = 8.'7; P


-2

~3

~5

>-9

~,)

lotal '

34 6

34 10 1.5

23 9 1.8

18 9 2.2

~ 9 3.5

2 9 5.4

0 6 0o

68 I¢~

xeld.i. = 12.86: P < IJ.01)l aMothers were classified as smokers if the), smoked more than one cigarette daily during the 3 Irimc'qers of pregnancy. Women were considered as non-smokers if they did not smoke the last trimester just before the beginning of pregnancy, if" they did not smoke during onc of the trimesters og" pregnancy or if they were not passive smokers. hTest for trend 117] calculated on non-cumulative distribution. CThis table represents the cumulative distribution level of AHH. (_'om,equently the this', >- 1 units inchided subjects of the class >~2 which included subject:-, in class >-3 and so on.

263 There was no evidence o f an association between A H H activity and placental m e a s u r e m e n t s i.e. weight, surface (assessed as largest d i a m e t e r × smallest diameter) a n d thickness. As generally o b s e r v e d in other studies, there was a consistent association between s m o k i n g and A H H activation in the samples (Table I11, test for trend: X21d.f = 12.8; P < 0.001). The same "threshold" p h e n o m e n o n observed a b o v e 5 units regarding placental calcifications and the level of placental A H H activity is also found here with s m o k i n g status and A H H activity. F u r t h e r m o r e , o u r d a t a showed that (Table IV) p a r e n c h y m a l calcifications are m o r e prevalent in the placenta o f smokers (Xeld.t -. = 7.5: P < 0.01) and to a lesser degree in basal plate (Xeld.~-. = 3.2: P < 0.07). The relative i r a p o r t a n c e o f the "smoking factor" and the A H H induction in their observed association with placental calcifications was analysed using a d j u s t m e n t by stratification [17]. When A H H activity was controlled, the association between s m o k i n g status and calcifications became non-significant (NS) (X2~d.t, = 0.6, NS; X21dt-, = 0.2, NS respectively for basal plate and parenchyma). W h e n s m o k i n g status was c o n t r o l l e d (Table 11), the association between placental calcifications and A H H i n d u c t i o n r e m a i n e d significant Xe)d~- = 5.5, P < 0.02: X-~ld.l-. = 5.6, P < 0.02 respectively for basal plate and parenchyma). It appeared that the association between s m o k i n g status d u r i n g pregnancy and calcifications o f the placental basal plate and the p a r e n c h y m a , previously observed, could be explained by the association o f these two factors with placental A H H induction. W h e n s m o k i n g status was c o n t r o l l e d using the detection o f cotinine in the m o t h e r ' s urine as an index o f t o b a c c o e x p o s u r e (Table I1), the association between placental calcifications and A H H i n d u c t i o n r e m a i n e d significant and thus unchanged (X21d.f = 9.1, P < 0.01; X21d.f. = 7.1, P < 0.01 respectively for basal plate and parenchyma).

TABLE IV DISTR|BUTION OF SMOKERS a ACCORDING TO THE EXISTENCE Ok PLACENTAL CALCIFICATIONS (BASAL PLATE AND PARENCHYMA) Calcifications

Smokers

Total

Basal plate

Parenchyma

No

Yes

No

Yes

Yes (16)

46 7

22 9

56 8

12 8

(84)

53

31

64

20

No (68)

X-Idf

=

3.2

P = 0.07

X-Idf

=

7.5

P < 0.01

"Mothers were considered as non-smokers if the~ did not smoke the last trimester just before the beginning of pregnancy, if they did not smoke during one of the trimesters of pregnancy or if they ~ere not passive smokers.

264

Discussion Smoking during pregnancy produces structural changes in human placenta, particularly in the basement membrane [19-22]. In addition, A H H activity increases as a consequence of maternal exposure to smoking. Thus, it appears that both the placental morphology and enzyme activities are altered by smoking during pregnancy. The results of the present study indicate that placental calcifications of both basal plate and parenchyma were associated with a marked increase in placental A H H activity. These findings were independently related to the smoking Tactor" as shown by the non-significance of the association between smoking status and calcifications when controlling for A H H activity. Therefore, the association between smoking and placental calcifications, previously documented [I l] and corroborated by the present study could be explained by A H H induction since, when allowing for cotinine in urine as index of smoking exposure, no change occurred in the initial level of significance. A major limitation of this study was our inability to measure the true exposure of the mothers to cigarette smoke since information regarding tobacco exposure was obtained by questionnaire. Mothers tend to underestimate the number of cigarettes smoked and the extent of smoke inhalation and/or may have had poor recall of their exposure to smoke. However, we were able to partially quantify the rate of potential misclassification by determining the levels of urinary cotinine in our subjects. The distribution of urinary cotinine among the mothers is shown in Table V. The subjects were divided into three classes. Class 1 grouped individuals with no detected cotinine, classes 2 and 3, of equal size, contained the others. Normally, one would expect to find an absence of cotinine in the urine of the 63 non-smokers and non-passive smokers. Yet 19% of these subjects turned out to be 'false' controls. It seems that some of the non-smokers were, either unknowingly exposed to passive smoke, or their exposure was too low to cause sufficient discomfort

TABLE V S M O K I N G STATUS A C C O R D I N G TO THREE CLASSES Ob C O T I N I N E IN MOTHER'S URINE (ng/mg of creatinine) G A T H E R E D AT DELIVERY Cotinine/creatinine (ng/mg)

0

1-84

Nonsmokers and non-passive smokers Passive smokers during the pregnancy (and non-smokers) Stopped smoking: before the beginning of pregnancy during the 1st trimester during the 2nd trimester Smokers during all stages of pregnancy

51

8

4

63

36

3

2

41

13 5 6 2 2

5 2 2 I 3

3 2 0 I 1I

2I 9 8 4 16

Note: potential misclassifications are in boldface and underscored.

>84

Total

265 for them to recall it. It is possible that others, given the high level of urinary cotinine detected (a level o f cotinine greater than 84 ng/mg o f creatinine was reported in 4 subjects), were smokers who concealed their tobacco habit out of a sense of guilt. In the other three groups (smokers t h r o u g h o u t pregnancy, smokers who stopped during pregnancy and passive smokers), it is much more difficult to evaluate the extent o f misclassification since the differences in the levels of urinary cotinine can be expected to vary substantially. One would, nonetheless, expect cotinine to be detected in all the smokers, yet this was not the case in 2 instances (12.5% of false smokers). It would have been possible to eliminate both the 'false controls" and the potential 'false smokers' from the study. Although this would have reduced the frequency of potential misclassification a m o n g smokers and the non-smokers groups, it would not have done so in the two other groups (passive smokers and smokers who quit during pregnancy). Also, it is possible to use the concentration of cotinine/creatinine in m o t h e r ' s urine as an index of tobacco exposure, but considering the relatively short biological half-life o f cotinine, it does not appear to be a reliable index in quantifying past tobacco exposure. In any event, it does not affect the associations. In order to treat all groups consistently we chose to accept a level of misclassification which would increase variance and therefore reduce the intensity of the observed relationships. Since calcifications were described [12] as one of the morphological hallmarks of placental aging often observed in normal pregnancies and merely associated with the normal aging o f the placenta [23], we could hypothesize that A H H activity was associated with a premature aging o f placenta. Nevertheless, it is difficult to reinforce this hypothesis with our data since gestational age was not associated with placental calcifications or A H H activity. If substantiated, the association between placental A H H induction and placental calcifications needs to be elucidated. Further studies could explore whether A H H activation was specifically and directly involved in placental calcifications, or whether A H H activation was the expression o f a much wider p h e n o m e n o n such as global increase o f enzyme activation. At this time it seems premature to propose plausible biochemical mechanisms to explain this association.

Acknowledgements This study has been supported by the French ministry of the environment (grant no. 86,316). We wish to acknowledge the staff o f the Baudelocque maternity clinic for its collaboration in obtaining specimens and Georgette Hellier and Josiane Sahuquillo for technical assistance.

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266 3 4 5 6

7 8 9 10 II 12 13

14 15 16 17 18 19 20 21 22 23

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