The effects of lead upon collagen synthesis and proline hydroxylation in the Swiss mouse 3T6 fibroblast

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

179,

15-23 (1977)

The Effects of Lead upon Collagen Synthesis and Proline Hydroxylation in the Swiss Mouse 3T6 Fibroblast DAVID

T. VISTICA,’

Departments of Molecular,

FRANKLIN

A. AHRENS,

AND

WARREN

R. ELLISON

Cellular and Developmental Biology and Veterinary Anatomy, Pharmacology Physiology, Iowa State University, Ames, Iowa 50011 Received May 24, 1976

and

The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular Fe2+/Pb2+ molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 N HClO, soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.

Increased capillary permeability in the cerebral cortex and cerebellum of suckling rats exposed to lead can be produced as early as 2 weeks postnatum without visible neurological involvement (1). Ultrastructural studies revealed that alterations in the collagenous component of the capillary basement membrane and intercellular junctions may be a primary event in the development of lead encephalopathy (2). As a result of this work we have proposed that interference in collagen metabolism by lead may be responsible for the failure of the lead-exposed cerebral microvasculature to regulate transcapillary flow. Because lead is known to antagonize other iron-requiring enzyme systems (e.g., ferrochelatase in heme biosyn-

thesis; 3-5) and because of the known dependency upon iron for the hydroxylation of proline (6-8) and lysine (9) during the initial stages of collagen biosynthesis, we investigated the effects of low levels of lead on the synthesis and turnover of collagen in the Swiss mouse 3T6 fibroblast. The results indicated that lead concentrations similar to those found in the cerebellum in lead encephalopathy (4-10 pg of Pb2+/g) produce alterations in the balance between collagen synthesis and degradation in 3T6, which may be attributed to interferences in the hydroxylation of proline. MATERIALS

AND METHODS

L-[“C]Proline (uniformly labeled, 290 mCi/ mmoll, L-[‘4Clhydroxyproline (uniformly labeled; 12 mCi/mmol), and L-[3,4-3H1proline (54 Ci/mmol) were purchased from Amersham Searle. Chromatographically purified collagenase was purchased from Worthington Biochemical Corp. and Dowex 5OW-X8 (200-400 mesh) was obtained from Sigma Chemical Co. The collagen-secreting 3T6 fibroblast was obtained from the American Type Culture Collection and minimal essential medium and fetal calf serum were purchased from Grand Island Biological Co. Cell culture. 3T6 cells were grown as previously described (10). Normally 3T6 cells are grown in Dul-

’ Presented to the Faculty of the Graduate School of Arts and Sciences of Iowa State University in partial fulfillment of the requirements for the Degree Doctor of Philosophy. Present address: Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. To whom correspondence should be sent. * Present address: School of Medicine, University of Iowa, Iowa City, Iowa 52240. 15

0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Copyright

ISSN 0003-9661

16

VISTICA,

AHRENS

becco-Vogts modification of Eagles medium (11) which contains iron in its formulation; however, in these studies it was desirable to examine the effect of low levels of lead on proline hydroxylation, and consequently, 3T6 was grown in MEM.3 Minimal essential medium contains no iron in its formulation but sufficient trace amounts were present to allow maximal hydroxylation of proline in lead-free cultures. Iron analysis of tissue culture medium components. Triplicate 20-ml samples of serum-free MEM, fetal calf serum, and MEM containing 10% fetal calf serum were dehydrated at 100°C for 3-4 h, ashed at 600°C for 5-8 h, and dissolved in 2 N HCl and iron concentrations were determined by atomic absorption spectrometry. Minimal essential medium contained 0.6 pglml of Fe*+ (10.7 PM), fetal calf serum contained 2.6 pg/ml of Fez+ (46.6 PM), and 90% MEM supplemented with 10% FCS contained 0.8 pg/ml of FeP+ (14.3 PM). Effect of lead upon 3T6 cell growth. Lead acetate (PbC,H,O,.3H,O; F.W. 379.33) was used in all studies described in this paper. 3T6 was seeded at a concentration of 1.4 x lo5 cells/ml in 25-cm2 Falcon flasks in the presence of 4.75 pglml of PbZ+ (22.9 PM; low dosage) or 7.0 kg/ml of Pb2+ (33.8 FM; high dosage). Replicate plates were trypsinized at specific intervals after seeding, and the cells were centrifuged and resuspended in Earle’s salt solution (ESS) containing trypan blue and counted on a hemocytometer. Cell viability was estimated by dye exclusion. Effect of lead on collagen synthesis, noncollagenous protein synthesis, and proline hydroxylation in 3T6 cells: The role of supplementary iron. All isotopic incubations were performed in 75-cm* Falcon flasks. Growth medium from stationary phase cultures was removed, and cells were rinsed three times with 10 ml of ESS and overlayed with 20 ml of serum-free MEM containing 50 ,ug/ml of freshly prepared ascorbic acid, 25 mM tricine (pH 7.4), and the low or high dosage of lead. In some experiments 1-15 pg/ml of iron was added. After a l-h incubation period, 5.6 &i of L-[‘*Clproline was added and the cells were incubated for an additional 120 min. The radioactive medium was removed and the cells were rinsed with two lo-ml portions of ESS. The rinses were added to the medium fraction, which was centrifuged at 15,000g (4°C) for 30 min to remove any cellular debris, dialyzed to background against 10 mM Tris.HCl (pH 7.6, 4”C), and lyophilized. Cells 3 Abbreviations medium; FCS, solution; TCA, phenyloxazole; zolyI)Jbenzene; tritiated water.

used: MEM, minimal essential fetal calf serum; ESS, Earle’s salt trichloroacetic acid; PPO, 2,5-diPOPOP, 1,4- bis[2- (5-phenyloxaBSA, bovine serum albumin; THG,

AND

ELLISON

were removed with 0.25% trypsin, immediately placed on ice, then rinsed three times with ESS (4’0, suspended in 1 ml of ESS, and sonicated at 20 W for lo-15 s on a Branson sonicator. Protein was precipitated by adding 1 ml of 10% TCA, the mixture was allowed to stand on ice for at least 1 h and then centrifuged at 60% to pellet cellular protein. Analysis of the culture medium and cell layers for collagen. The procedures used for purification of collagenase on Sephadex G-200, determination of its degree of purity, and its use in digesting heterogeneous protein mixtures containing collagen have been described (10, 12). Protein was determined by the method of Lowry et al. (13). All radioactive samples were counted in 15 ml of liquid scintillation cocktail (toluene; Triton X-100,2:1, v/v containing 4 g/liter of PPO and 50 mglliter of POPOP). Analysis of collagenase-derived peptides for proline and hydroxyproline. Hydrolyzed samples were evaporated to dryness in a gentle stream of air and proline and hydroxyproline were analyzed either by Chloramine T oxidation using recent modifications (12) of the original method (14) or by separation on Dowex 5OW-X8 columns using citrate buffer (0.1 N) as an eluant (15). Recoveries were calculated from standards containing known amounts of L1’4C1proline and L-[14C1hydroxyproline. Collagen breakdown experiments. 3T6 was seeded at 1.2 to 1.5 x lo” cells/ml in complete medium and allowed to complete lag phase (19 h). Ascorbic acid (50 pg/ml) was added to all cultures and the low or high dosage of lead was added to all but control cultures of 3T6, which received an equivalent volume of ESS. After a l-h equilibration period, 5.6 &i of L-lL4C1proline was added to each culture. Medium from terminal logarithmic phase (42 h postseeding) cultures was removed and was centrifuged at 15,000g to remove cellular debris. The cell layer was removed with 0.25% trypsin, rinsed three times in ESS, suspended in 1 ml of ESS, and sonicated. An aliquot was removed for protein determination, and both the cell layer and medium were made 0.5 N in perchloric acid and allowed to stand 12 h on ice (16). Soluble medium and cell layer fractions were neutralized with 16 M KOH, made 6 N in HCl, and hydrolyzed (16 h, 15-20 psi, 120°C). Insoluble medium and cell layer fractions were suspended in 6 N HCl and hydrolyzed. Hydrolyzed samples were decolorized using a charcoal-resin mixture (17) and evaporated to dryness. Hydroxyproline was isolated on Dowex 50 columns. Kinetic studies: Proline hydroxylase assays. Substrate for proline hydroxylase assays was prepared according to previously published methods (18-20). Proline hydroxylase assays were performed according to a previously described method (21) except that the concentration of Tris’HCl buffer was reduced from 200 to 100 kmol, and 2 mg of boiled

LEAD AND COLLAGEN bovine serum albumin (BSA) was added. (22). The complete reaction mixture consisted OE Tris.HCl, pH 7.5, 100 pmol; a-ketoglutarate, 0.2 pmol; Fe(NH,),(SO,),.6H,O, 0.5 to 50 Fmol; boiled BSA, 2 mg; ascorbic acid, 1.0 pmol; t[3,4JH]proline chick embryo substrate, 740 pg, 221,200 dpm; 3- to 5-dayold confluent 3T6 cell sonicate, 300 pg; water and/or varying-concentrations of Pb*+, 0.5 to 50 pmol; contained in a total volume of 2.0 ml. The reaction was started by addition of the 3T6 cell sonicate and stopped by addition of 0.22 ml of 50% TCA. Reaction mixtures to which TCA was added before the sonicate served as controls. Tritiated water (THO) derived from these blanks was subtracted from all experimental values. Tritiated water was collected by vacuum distillation (20) of 2.0-ml aliquots of the reaction mixture. Distillate was transferred into tared scintillation vials and weighed. Fifteen milliliters of scintillation fluor was added and samples were counted for 100 min (approximately 1.5% counting error) on a Beckman LS-100 spectrometer. Data were corrected for the portion of the reaction mixture that was not distilled (0.22 ml) and for slight variation in recovery at the distillation step of the assay. RESULTS

Administration of lead at the time of seeding of 3T6 cells resulted in a significant effect on cell growth (Fig. 1) without

Hours

FIG. 1. 3T6 was seeded in 25cm” Falcon flasks at a concentration of 1.4 X lo5 cells/ml (7.0 X lo5 cells/ plate) in the presence of 22.9 PM Pb*+ or 33.8 &M Pb*+. Control cultures received an equivalent volume of ESS. Cells were removed from replicate plates at indicated times by trypsinization, suspended in ESS containing trypan blue, and counted on a hemocytometer. Values represent means derived from each sampling period.

SYNTHESIS

17

producing cellular degeneration or death as suggested by exclusion of trypan blue. The total cell number of terminal logarithmic phase cultures (42 h) receiving the low or high lead dosage level was decreased by approximately 50%. Addition of lead to 3T6 cultures resulted in stimulation of incorporation of L-[14C]proline into the cell layer (Table I). Cultures receiving lead exhibited increases in total radioactivity which was accompanied by decreases in the secretion of radioactively labeled macromolecules into the culture medium. Addition of lead resulted in increased collagen and noncollagenous protein in the cell layer. Similar stimulation of protein synthesis by lead has been described by Choie and Richter (23) in kidney proximal tubular epithelium. Lead-exposed cultures exhibit increases in cell layer-associated collagen and decreases in secretion of collagen and noncollagenous protein into the medium. The increase in collagen associated with the cell layer corresponds closely with the decrease in the secretion of collagen into the medium. Impaired hydroxylation of cell layer-associated collagen in 3T6 cultures exposed to lead was evident by increases in the proline/hydroxyproline ratio of collagen polypeptides (Table II). Although the amount of collagen secreted by 3T6 cultures exposed to lead is reduced, the collagen found in the culture medium is fully hydroxylated. Addition of iron to 3T6 cultures incubated in the low or high dosage levels of lead resulted in a substantial protective effect, which increased as the extracellular FeZ+/Pb2+ molar ratio increased; complete protection occurred at a molar ratio of about 5.0 (Table III). Increases in the extracellular Fe2+/Pb2+ molar ratios resulted in a decrease in cell layer-associated unhydroxylated procollagen, which can be attributed to partial protection by iron of lead-induced inhibition of proline hydroxylation. Breakdown of collagen occurs during logarithmic phase of growth of control 3T6 cultures (Table IV). It is evident that control cultures of 3T6 accumulate significant amounts of acid-soluble hydroxypro-

73,228 94,819 113,262

Cell layer

Total

8,587 5,802 3,532

Medium 2,737 3,655 3,950

Cell layer

Collagen

2,830 1,875 1,606

Medium

(dpmlmg protein)

HYDROXYLATION,

I

5,567 5,530 5,556

Total

of cell

PROTEIN

70,506 91,067 109;306

Cell layer 5,720 3,927 1,926

Medium

76,226 94.994 1111232

Total

(dpml

SYNTHESIS

Noncollagenous protein mg of cell protein)

AND NONCOLLAGENOUS

TABLE

cells were pulsed for 120 min with 5.6 &i of [W]proline in serum-free medium and Methods. Values represent means derived from three separate experiments.

81,815 100,621 116,794

of

PROLINE

Total

radioactivity (dpmlmg cell protein)

SYNTHESIS,

’ Five-day-old stationary phase processed as described in Materials

Control 22.9 /.m Pb*+ 33.8 /.LM Pb2+

Culture des&nation

EFFECTS OF LEAD ON COLLAGEN

1,350 890 747

Medium

2,656 1,599 1,377

Total

of

and cell layers and medium were No Fez+ was added to cultures.

1,306 709 630

Cell layer

Hydroxyproline (dpm/mg cell protein)

IN 3T6 FIBROBLASTV

z

s 8

5 cn

$

’

J 2

LEAD

AND COLLAGEN TABLE

HYDBOXYLATION

Ctrkr;t;s-

OF PROJJNE

RESIDUES

II

Collagen proline (dpm/mg of cell protein)

Collagen hydroxyproline (dpmlmg of cell protein)

Cell layer

Cell layer

Cell layer

Medium

3T6 PEPTIDES’ Collagen proRelative hydroxylation (%) lin;ffgw

IN COLLAGENASE-DERIVED

Co11 en (dpm/ mg ?0~2:; pro-

Medium

19

SYNTHESIS

Medium

Cell layer

Medium

Cell layer

Medium

Controlb 2650 2850 1406 1492 1245 1350 1.13 1.11 100 100 22.9 /.m Pb*+ 3600 1830 2823 958 692 865 4.17 1.11 40.95 100 33.8 /AM Pb2+ 3926 1560 3292 825 632 738 5.21 1.12 34.30 100 a Five-day-old stationary phase cells were pulsed for 120 min with 5.6 &i of [Wlproline in serum-free medium and the cell layers and medium were processed as described in Materials and Methods. Peptides derived from collagenase digestions were analyzed for extent of hydroxylation. Values represent means derived from two separate experiments. No Fez+ was added to cultures. b Control culture is designated as being fully hydroxylated; cultures receiving lead are compared to controls. TABLE EFFECT

OF ADDITION

OF IRON

Fez+a (@I)

Pb2+ (PM)

Fe2+/Pb2+ (PM)

10.7 30.0 62.0 135.0 121.0 270.0 243.0

0 22.9 22.9 33.8 22.9 33.8 22.9

Control 1.3 2.7 4.0 5.3 8.0 10.6

UPON PROLINE

Cell layer collagen (dpm/mg of cell protein) 2956 3206 3107 3075 3025 2896 2850

III

HYDROXYLATION

Coll;ren ($m/mg of cell protein) 1564 2004 1880 1774 1606 1550 1520

IN 3T6 CULTURES

EXPOSED

TO LEAD

Collagen hydroxyproline (dpmlmg of cell protein)

Collagen pro-

Hydroxxylation 0

1360 1196 1228 1292 1401 1340 1325

1.15 1.68 1.53 1.37 1.15 1.16 1.15

100.0 80.2 85.0 90.4 100.0

99.6 100.0

a Iron was added to 3T6 cultures in addition to 22.9 or 33.8 FM Pb2+. The iron concentrations listed include the 10.7 PM Fez+ endogenous to serum-free MEM. Five-day-old stationary phase 3T6 cultures were pulsed for 120 min with 5.6 &i of [Wlproline in serum-free medium and the peptides derived from collagenase digestion were analyzed for extent of hydroxylation. Proline/hydroxyproline ratios for cultures receiving 22.9 or 33.8 PM Pb*+ and no supplementary iron were similar to those described in Table II. The values were derived from a single experiment. TABLE EFFECT

OF LEAD

Culture desig-

UPON COLLAGEN

Hydroxyproline (dpm/mg of cell

nation

Insoluble

BREAKDOWN

Medium Total

Soluble

IN

3T6 CELLS” Soluble hydroxyprolinelinsoluble hydroxyproline

protein)

Cell layer Soluble

IV

Insoluble

Cell

layer

Medium

Soluble hydroxy-

proline (%I Cell

layer

Medium

Total

Control 1,936 6,943 8,879 33,107 33,290 66,397 0.28 0.99 21.80 49.9 22.9 PM PbZ+ 2,221 7,298 9,519 32,612 30,836 63,448 0.30 1.06 23.30 51.4 33.8 /LM Pb2+ 2,377 6,158 8,538 31,280 25,182 56,462 0.3Sb 1.24' 27.90 55.4 a 3T6 cultures were pulsed for 23 h with 5.6 &i of [‘4C]proline during logarithmic phase of growth; cell layers and medium were made 0.5 N in perchloric acid and processed as described in Materials and Methods. No Fe*+ was added to cultures. Values represent means derived from two experiments. b Statistically significant from control cultures by Student’s t test at P < 0.1. c Statistically significant from control cultures (P < 0.05) and cultures receiving 22.9 pM Pb*+ (P < 0.1).

20

VISTICA,

AHRENS

line. Enhancement of collagen breakdown occurs in 3T6 cultures exposed to lead as reflected by increases in the 0.5 N HClO, soluble/insoluble hydroxyproline ratio in both the cell layer and medium (Table IV). Since hydroxyproline cannot be reutilized for new collagen synthesis (24), these results suggest that interference with the hydroxylation of proline by lead (Table II) increases the rate of breakdown of partially hydroxylated collagen. It is probable that the presence of significant amounts of iron in the growth medium reduces the magnitude of the effect produced by lead. The appearance of increased amounts of acid-soluble hydroxyproline in the culture medium of cells exposed to lead indicates that 3T6 may be able to secrete these breakdown products into the medium. Since addition of iron to 3T6 cultures exposed to lead resulted in protection from lead-induced inhibition of proline hydroxylation (Table III), the kinetic mechanism was investigated. The sonicate exhibited dependence upon iron for the hydroxylation reaction; omission of iron from the assay mixture resulted in tritiated water release which was equivalent to control values. A time-course reaction was run in order to assure that experiments were carried out under conditions of initial velocity. Under these experimental conditions reaction velocity, as measured by the release of THO, was constant over the period of assay for any given concentration of iron. The time course was run at saturating iron concentrations, and the results indicate that the release of THO is linear with time for approximately 10 min (Fig. 2). On the basis of this determination, subsequent experiments were run with comparable cell sonicate concentrations for 10 min. The results of the kinetic analysis indicate that the mechanism is probably competitive (Fig. 3). Increasing concentrations of iron were accompanied by increased hydroxylation of proline residues in the presence of lead; in addition, identical apparent maximal velocities were obtained. A replot of the slopes of the Lineweaver-Burke plot (Fig. 3) against lead concentration is linear (Fig. 4).

AND ELLISON

Minutes

FIG. 2. Time course for release of tritiated water (THO) from n-13, 4-3H1proline chick embryo protein substrate using 3T6 sonicate as the source of proline hydroxylase. The reaction mixture contained 884,800 dpm of 13, 4-3H]proline substrate, 0.1 mM (Yketoglutarate, 0.1 mM Fee+, 0.5 mM ascorbic acid, 1 mglml of boiled bovine serum albumin, 0.4 ml of H,O, and 50 mM Tris.HCl (pH 7.5) in a total volume of 7.4 ml. The reaction was started by addition of 1200 pg of 5-day-old 3T6 sonicate. One-milliliter aliquots were removed at 5, 10, l&20,30,45, and 60 min, 0.11 ml of 50% TCA was added to stop the reaction, and THO was collected by vacuum distillation.

015

1.0 1 /Fe’*x

TN5

2b

lo6

FIG. 3. Proline hydroxylase assay using varying iron concentrations at two levels of lead. Each assay tube contained the standard reaction mixture in a total volume of 2.0 ml: 221,200 dpm of chick embryo substrate, 100 pmol of Tris.HCl (pH 7.5), 0.2 pmol of a-ketoglutarate, 2 mg of boiled bovine serum albumin, 1.0 pm01 of ascorbic acid, Fe*+ (0.5, 0.714, 1.25, or 5.0 ~moll, and either Pb*+ (1.0 or 3.0 pmol) or an equivalent volume of water. The reaction was started by addition of 300 Fg of B-day-old 3T6 sonicate and stopped after 10 min by addition of 0.22 ml of 50% TCA. THO was collected by vacuum distillation.

LEAD

I 1.15 KPb2+

1.0 Micromoles

2.0 Pb*’

AND

COLLAGEN

30

FIG. 4. Replot of slopes derived from Fig. 3 against Pb2+ concentration in proline hydroxylase assay. Extrapolation back to the abscissa gives an apparent inhibition constant, Kpb*+ of 1.15 pmol. DISCUSSION

Susceptibility to lead toxicity is enhanced by a number of environmental factors including age, decreased calcium and phosphorous intake, iron deficiency, various heavy metals (e.g., cadmium), and increased intake of vitamin D (25). Ascorbic acid was reported to be beneficial in preventing neurological symptoms of lead encephalopathy (26); however, these results were not confirmed by subsequent studies (27, 28). The increased severity of lead toxicity which occurs under conditions of iron deficiency (29) is apparently the result of increased absorption and transport of lead, resulting in increased content of lead in liver, kidney, and bone. A similar situation may occur in the cerebellum during the development of lead encephalopathy. The increases in lead concentration in the cerebellum of 2- to 4-week-old suckling rats exposed to lead, described by Goldstein et al. (30), suggest that increased transport of lead by the capillary endothelial cell occurs and may be responsible for the observed effects of lead upon the collagenous component of the basement membrane (2). Little work has been reported on the effects of lead on collagen metabolism. Hass et al. (31) speculated that the mechanism of vascularly related lead toxicity may reside in its effect upon connective tissue metabolism. Specifically, these authors found that lead subacetate inhibited collagen formation in rabbits under conditions of elevated vitamin D intake. The effects of lead upon protein synthesis in 3T6 cells (Table I) reveal that lead

SYNTHESIS

21

impairs the secretion of both collagen and noncollagenous protein under conditions which approximate cerebellar tissue lead concentrations during the development of lead encephalopathy (4-10 pg of Pb2+/g). These results also suggest that lead produces a depression in the relative rate of collagen biosynthesis since incorporation of proline into collagen remained constant in cells exposed to 22.9 or 33.8 PM lead, while proline incorporation into noncollagenous protein increased by 24.5 and 46%, respectively. It is also apparent that proline is significantly underhydroxylated under these conditions (Table I and II), resulting in increased retention of unhydroxylated procollagen. It has generally been accepted that proper hydroxylation of the proline residues in collagen is necessary if optimal secretory rates are to be maintained. Autoradiography revealed that inhibition of proline hydroxylation results in cellular retention of unhydroxylated procollagen (32). However, the fate of unhydroxylated procollagen produced under conditions in which one or more of the cofactors required for proline hydroxylation (Fez+, ascorbic acid) are 02, a-ketoglutarate, limiting, apparently is dependent upon cell type. Two commonly used methods to produce accumulation of unhydroxylated procollagen are ascorbate deficiency and chelation of iron with a,&-dipyridyl. Ascorbate deficiency in Balb 3T6 fibroblasts and chick embryo fibroblasts (33) results in significantly reduced secretion of unhydroxylated procollagen. In addition, total collagen synthesis is reduced in ascorbate deficient chick embryo fibroblasts. Incorporation of [14Clproline into collagenous and noncollagenous protein is not altered in ascorbate-deficient cultures of 3T6 (34). Ascorbate deficiency in this cell line results in secretion of significant amounts of unhydroxylated procollagen (35); in addition, the molecular weight and the amount of collagen secreted into the culture medium do not differ from that of ascorbate supplemented cell cultures (36). There is considerable evidence that reduction in the hydroxyproline content of both synthetic peptides (37) and of chick tendon collagen

22

VISTICA,

AHRENS

(38, 39) results in a decrease in thermal stability and also to increased susceptibility to tissue protease (40) and pepsin (41) digestion. These observations suggest that association of pro-a chains ‘into a triple-helical conformation is at least partially dependent upon hydroxyproline content. It is possible that unhydroxylated procollagen produced during administration of lead to 3T6 cultures is nonhelical in nature. It appears that the significant reduction in hydroxylation of collagen proline residues (Table II) and the observed increases in acid-soluble hydroxyproline (Table IV) are interrelated. Failure of lead-induced unhydroxylated procollagen to assume triple-helical conformation would result in increased intracellular susceptibility to protease degradation. Increases in the 0.5 N HClO, soluble/insoluble hydroxyproline ratio in 3T6 cultures exposed to lead (Table IV) suggest that interference with the hydroxylation of proline results in an increase in the rate of breakdown of partially hydroxylated collagen. Unhydroxylated procollagen synthesized by logarithmic phase cultures of 3T6 cells (35) has been shown to be more susceptible to breakdown than fully hydroxylated collagen (16). Recent work in this laboratory and by Blanck and Peterkofsky (42) with (~,a’dipyridyl indicates that there is a correlation between the amount of collagen appearing in the culture medium and its degree of hydroxylation. The ability of supplementary iron to protect 3T6 cells from inhibition of proline hydroxylation by lead (Table III) and the apparent competitive kinetics of the interaction (Fig. 3) indicate that lead and iron are competing for the same site on proline hydroxylase. The observation that the replot (Fig. 4) of the Lineweaver-Burke (Fig. 3) is linear indicates simple competition between lead and iron for the active site of proline hydroxylase. On the basis of these kinetic data (identical apparent maximal velocities and a linear replot), it appears that lead does not bind to sites on the enzyme (e.g., to SH groups) other than the active sites.

AND ELLISON

The increased permeability of cerebellar capillaries in lead encephalopathy in rats is associated with impaired development of capillary basement membrane (2). Whether lead acts by inhibition of synthesis or enhanced degradation of collagen in the basement membrane is unknown. However, in 3T6 cultures, inhibition of proline hydroxylation decreases the amount of collagen secreted by this cell and may impair the association of collagen into a triple-helical conformation. Thus, interference with this initial unique stage in collagen biosynthesis results in a molecule with increased susceptibility to intracellular degradation. Investigation of the relative rates of turnover of collagen in brain capillary basement membrane should provide further insight into the mechanism of lead-induced encephalopathy. ACKNOWLEDGMENTS This study was supported by Grant ES00633 from the National Institute of Environmental Health Sciences, National Institute of Health. We thank Dr. Jeanine Carithers (Department of Veterinary Anatomy, Pharmacology and Physiology, Iowa State University) and Drs. Marco Rabinovitz and Beverly Peterkofsky (National Cancer Institute) for review of the manuscript. REFERENCES 1. AHRENS, F. A., AND VISTICA, D. T. (1977) Exp. Mol. Pathol., in press. 2. VISWA, D. T., AND AHRENS, F. A. (1977) Exp. Mol. Pathol., in press. 3. RIMINGTON, C. (1937) Comp. Rend. Lab. CarZsberg Ser. Chim. 22, 454-464. 4. JANDL, J. H., INMAN, J. K., SIMMONS, R. L., AND ALLEN, D. W. (1959) J. Clin. Invest. 38, 161-

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Anal. Biochem. 4, 400-406. 15. KLEIN, L., FAULKNER, W. R., AND ARMANDA, D. P. (1971)Sfund. Meth. Clin. Chem. 6,41-56. 16. STEINBERG, J. (1973) J. Cell Sci. 12, 217-234. 17. PROCKOP, D. J., AND UDENFRIEND, S. (1960) Anal. Biochem. 1, 228-239. 18. PROCKOP, D. J., AND JUVA, K. (1965) Proc. Nut. Acad. Sci. USA 53, 661-668. 19. HUWON, J. J., AND UDENFRIEND, S. (1966) Proc. Nat. Acad. Sci. USA 56, 198-202. 20. HURON, J. J., TAPPEL, A. L., AND UDENFRIEND, S. (1966) Anal. Biochem. 16, 384-394. 21. HUWON, J. J., TAPPEL, A. L., AND UDENFRIEND, S. (1967) Arch. B&hem. Biophys. 118, 231240. 22. GRIBBLE, J. T., COMSTOCK, J. P., AND UDENFRIEND, S. (1969) Arch. B&hem. Biophys.

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