In vivo formation of hydroxyl radicals following intragastric administration of ferrous salt in rats

In Vivo Formation of Hydroxyl Radicals Following Intragastric Administration of Ferrous Salt in Rats Jae 0. Kang, Adam Slivka, Gary Slater, and Gerald Cohen Departments of Neurology and Surgery, and Graduate School ofBiomedical Sciences, Mount Sinai School of Medicine and City University of New York, New York, New York

ABSTRACT Accidental poisoning by oral iron preparations is a serious problem in young children. We investigated the formation of hydroxyl radicals (*OH) in rats after intragastric instillation of ferrous sulfate. *OH was detected via its reaction with intragastrically administered 2-keto4-methylthiobutyrate to generate ethylene gas. Ascorbic acid is typically present in oral iron preparations in order to facilitate absorption by maintaining iron in the reduced state. However, ascorbate possesses two properties that can affect *OH: recycling of oxidized iron to the ferrous state augments *OH production, while ascorbate in high concentration scavenges *OH. In experiments conducted in vitro, both actions were evident, depending upon the concentration of ascorbate. In parallel experiments conducted in viv’o, the scavenging action of ascorbate was more prominent. Experiments in vitro with *OH-scavengers (dimethylsulfoxide, ethanol) and with the enzyme, catalase, confirmed both the presence of *OH and its dependence upon generated hydrogen peroxide during the oxidation of ferrous salt by molecular oxygen. Hydroxyl radicals (and/or reactive higher oxidation states of iron) may play a role in tissue damage after accidental overdose of oral iron.

INTRODUCTION poisoning by oral iron preparations is a major cause of accidental death in young children [ 11. A primary finding at autopsy in both adults and children is severe hemorrhagic necrosis of the stomach and proximal small bowel [2-51. Similar findings have been reported in experimental animals given high oral doses of iron salts [6, 71. The mechanisms for the toxicity of oral iron are not understood. We report here on Accidental

Address reprint requests to Dr. Gerald Cohen (Anbg. 14-70), Dept. Neurology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. Journal

of Inorganic

Biochemistry

35, 55-69

(1989)

@ 1989 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas,

55

NY, NY 10010

0162-0134/89/$3.50

56 J. 0. Kang et al.

the formation of a highly destructive free radical, the hydroxyl radical (-OH), after gastric instillation of ferrous sulfate in rats. Ever growing evidence supports the view that *OH plays an important role in vivo in mediating aspects of cellular pathology [8, 93. In vitro, *OH attacks a variety of biomolecules, including DNA, proteins, polyunsaturated fatty acids, and polysaccharides [8, 10-131. We suggest that *OH may be responsible, in part, for damage to the gastrointestinal tract in poisoning by oral iron, and that it may share responsibility, as well, for the gastrointestinal distress of persons who exhibit sensitivity to oral iron. Oral iron is widely prescribed to treat anemia, and it is routinely taken by many women to ward off anemia during menstrual bleeding. Most oral iron preparations consist of a ferrous salt, rather than a ferric salt, as the source of elemental iron [l, 141. The reason is that iron is absorbed from the gastrointestinal tract most efficiently when it is in the ferrous form. Many iron preparations additionally contain ascorbic acid in order to maintain the iron in the reduced state. Ferrous ions and certain ferrous chelates (e.g., ferrous-EDTA) in solution are readily oxidized by molecular oxygen. The redox reactions of iron and oxygen generate a number of reactive forms of reduced oxygen. Under acidic conditions, such as those present in the stomach, the reactive species are the hydrodioxyl radical (*OOH, Eq. l), hydrogen peroxide (H202, Eq. 2), and the hydroxyl radical (-OH, Eq. 3). Similar reactions occur even more vigorously at cellular pH, except that under neutral conditions, the hydrodioxyl radical exists in its ionized form, the superoxide radical (0~~; Eq. 4). Fez+ +0,+H++Fe3+

+ *OOH

-0OH -I- *00H+02 Fez+ +HZOzfH++Fe3+ .00H-r02-

(1)

+ HzOz

(2)

+ *OH+H,O

(3)

+H+

(4)

The generation of - OH from Fe *+ via the sequential reactions shown in Eqs. l-3 can be prolonged when Fe*+ is regenerated by recycling iron from the oxidized to the reduced state by strong reducing agents such as ascorbic acid (Eq. 5, where AH2 is ascorbic acid, and A is dehydroascorbic acid) [15]. Whereas -0OH is a strong oxidant [16], superoxide, with which it is in equilibrium, is a good reductant; therefore, at neutral pH, iron can be recycled, as well, by superoxide (Eq. 6) [15, 171. It is important to note, however, that ascorbic acid can also react directly with *OH (Eq. 7, where *AH is the monodehydroascorbate radical). When present in high concentration, ascorbic acid becomes an excellent scavenger for -OH. 2Fe3+ +AH2-r2Fe2+ +A+2H+ Fe3+02--Fe*+

+0

.OH+AH,+-AH+H,O

2

(5) (6)

(7)

The data presented here extend a preliminary report [ 181 on -OH formation by intragastric ferrous salt. We have now examined dose-response relationships for the generation of -OH by oral iron in rats, and the dose-response effects of ascorbic acid. Parallel experiments conducted in vitro reveal differences in the action of ascorbic acid in vivo and in vitro. In addition, we have examined the effects of *OH scavengers in

HYDROXYL

RADICALS IN VIVO FROM ORAL IRON

57

vitro and provide evidence that the mechanism for *OH production is a Fenton reaction between ferrous ions and generated hydrogen peroxide. These observations may set the stage for new ways to intervene in iron-mediated toxicity. METHODS Materials

The sodium salt of 2-keto-4-methylthiobutyric acid (2-keto-4-methiolbutyric acid, KMB), urea, and diethylenetriaminepentaacetic acid (DTPA) were purchased from Sigma Chemical Co. (St. Louis, MO); ferrous sulfate (FeSOd-7H20), hydrogen peroxide (30%), L-ascorbic acid and dimethylsulfoxide (M%SO) were from Fisher Scientific Co. (Fair Lawn, NJ); 95% ethanol from Publicker Chemical Co. (Philadelphia, PA); catalase (crystalline aqueous suspension, 74,600 units/mg) from Worthington Biochemicals (Freehold, NJ); and bovine albumin Fraction V from Nutritional Biochemical Corp. (Cleveland, OH). Hydrocarbon-free (zero grade) air, oxygen, helium, and hydrogen were obtained from Linde (South Plainfield, NJ) and ethylene calibration standard from Alltech Associate (Deerfield, IL). Statistical comparisons were carried out by 2-tailed Student’s t-test. Animals Female Sprague-Dawley rats (Perfection Breeders, Douglasville, PA) weighing 220250 g were fed Purina lab chow. In an initial set of experiments, rats were fed ad libitum. In all other experiments, food was withheld for 18 hr beforehand, but animals were allowed free access to water. Experiments were initiated between 9:30-11:00 A.M.

Detection System for Hydroxyl Radicals

The generation of -OH was monitored by measuring ethylene gas formed in the presence of KMB, which reacts avidly with *OH and generates ethylene as a product (Eq. 8) [19, 201. The ethylene was measured by gas chromatography. CH3-S-CH2-CHz-CO-COOH

+ *OH -*CH2 = CH2 + other products

(8)

Gas Chromatography Samples (30-50 ml) of chamber air from in vivo experiments were analyzed for ethylene with a Hewlett-Packard Model 5750 gas chromatograph [21]. Briefly, a charcoal precolumn in conjunction with a Chemical Data Systems Model 310 concentrator (Oxford, PA) was used to trap the ethylene, which was subsequently flash desorbed directly onto the gas chromatographic column. Analyses for ethylene were conducted with a 1.8 m stainless-steel column that was packed with Porapak N (60180 mesh). A flame ionization detector was used. For in vitro experiments, the charcoal precolumn and concentrator were bypassed, and the 0.5 ml vol was injected directly. Ethylene was quantitated from the peak height by comparison with known standards. In Vivo Experiments All solutions were prepared in deionized, distilled water that was polished with a

Millipore Q system (Millipore Corp.). Solutions were prepared immediately before use in cold distilled water in an ice bath by homogenization in glass tubes with teflon

58

J. 0. Kang et al.

grinders. This procedure produced ferrous sulfate solutions that exhibited some turbidity; no settling occurred during the time required for injections. The solutions were loaded into l-ml or 34 plastic syringes and were injected into the gastric cavity via a stainless steel feeding needle. The feeding needle volume was 0.2 ml. The gastric needle was first flushed and filled with distilled water, and then it was inserted into the rat with an attached syringe filled with KMB solution. Injections were made rapidly and sequentially by exchanging syringes, while the needle remained in place in the rat. KMB injection was followed either by ascorbate and then iron (Table 1) or by a freshly prepared mixture of ascorbate and iron (all other experiments). The last syringe contained 0.5 ml of water as a rinse. Control rats received KMB followed by a volume of water that encompassed all other volumes injected into experimental rats. Immediately after intragastric instillations, the animals were placed into breath collection chambers. Methodological details of this system are described by Lawrence and Cohen [21]. Each chamber with attached tubing and accessories was first flushed with room air for 30 min by opening the outlet and turning on the pump. Subsequently, the chamber was purged with hydrocarbon-free air for 5 min, and the rat was inserted. The chamber was then sealed, and the enclosed atmosphere was continuously circulated by means of a pump. The system contained traps for exhaled carbon dioxide and ammonia; hydrocarbon-free oxygen was drawn in from an outside supply to replace the carbon dioxide as it was removed. The animals did not show any signs of distress following the injections or during subsequent monitoring of exhaled ethylene. The first sample (30-50 ml) was withdrawn after 5 min and was used as a zero time value. Subsequent samples were withdrawn as indicated in the text.

TABLE 1. Production of Ethylene by Rats after Intragastric Instillation of KMB with and without Ascorbic Acid Plus Ferrous Sulfate0 Ethylene Production

KMB + Iron + Ascorbic Acid

KMB Alone Time (hr)

nmollkg 0.3 * 0.3 2.4 4.1 5.9 7.9 9.4 10.5 10.5

+ * i + * + +

0.3 1.0 0.8 0.8 1.1 2.4 2.4

nmol/kg *hr 0.3 2.1 1.7 1.8 2.0 1.5 1.1 0.0

nmol/kg 17.0 45.6 70.1 104.0 140.0 165.4 177.3 174.8

* f f * f f f f

7.4 21.7 31.7 45.0 71.2 80.5 91.2 84.6

nmollkg *hr 17.0 28.6 24.5 33.9 36.0 25.4 11.9 (-) 2.5

’ Sprague-Dawley rats, fed ad libitum, received sequential gastric instillations of 0.5~ml vol of distilled water containing KMB (100 mg), L-ascorbic acid (100 mg), and FeS04.7H20 (100 mg), followed by a rinse with 0.5 ml of water. Samples of air from the sealed chambers were analyzed for ethylene by gas chromatography. The cumulative production of ethylene and the rate for each hourly interval are shown. Data are the mean f SD for n = 4, except n = 3 for KMB alone at 6,7, and 8 hr. The differences between rata treated with KMB alone and those treated with KMB + ascorbic acid + iron were statistically significant: p < 0.01 at 1, 2, 3, and 4 hr, and p < 0.025 at 6, 7 and 8 hr.

HYDROXYL

RADICALS

IN VIVO FROM ORAL IRON

59

In Vitro Experiments In vitro studies were carried out in 25-ml Erlenmeyer flasks. The flasks were sealed with rubber septa that permitted sampling of the atmosphere by syringe to assess the presence and amount of ethylene. Reaction mixtures with appropriate components were prepared in a final volume of 2.5 ml. Ferrous sulfate was added last to initiate the reactions. The flasks were incubated in a shaking water bath at 37°C. The medium was 4 mM HCl, except for experiments with catalase, where water was used. Headspace air samples (0.5 ml) were removed and analyzed by gas chromatography. The crystalline catalase suspension was centrifuged at 700 x g in a refrigerated centrifuge. The isolated crystals were washed three times by resuspension in ice-cold distilled water, followed by recentrifugation, in order to remove thymol present in the commercial enzyme preparation. A stock solution was prepared at 2 mg/ml. Distilled water was used as the medium in order to limit the inactivation of enzymatic activity; 4 mM HCl inactivated catalase. Heat inactivation was achieved by placing the stock catalase in a boiling water bath for 15 min; this procedure resulted in loss of > 99% of the activity as determined by the spectrophotometric assay of Luck [22]. Denatured protein was triturated several times through a 20-gauge needle. Bovine serum albumin at a concentration of 2 mg/ml was used as a protein control. Because catalase in water is inactivated by high concentrations of ferrous sulfate and KMB, it was necessary to work at lower concentrations of these reagents. The final reaction mixtures contained 2.4 mM KMB, 0.2 mM L-ascorbic acid, 0.48 mM FeS04, and 0.7 mg protein in 2.5 ml water.

RESULTS In Vivo Production of Ethylene by Rats after Intragastric Administration of Ferrous Sulfate Table 1 shows a time course for ethylene production after intragastric instillations of KMB, ferrous sulfate, and ascorbic acid in rats that had been fed ad libitum. In all subsequent experiments, food was withdrawn 18 hr beforehand, and measurements were initiated between 9:30-l 1:00 A.M., in order to limit experimental variables. The effect of ascorbate on ethylene production was studied in separate experiments (see ahead). In Table 1, animals receiving KMB alone produced small amounts of ethylene, while rats receiving KMB followed by ascorbate and iron produced very much larger amounts. Control rates of ethylene production were less than 2.1 nmol/kg/hr over the 8-hr experimental period. Experimental animals showed a peak hourly rate of 36.0 nmol/kg/hr. Ethylene production ceased after 7 hr. Neither iron alone nor KMB plus ascorbic acid produced any more ethylene than KMB alone (data not shown). Figure 1 shows ethylene production after intragastric instillation of 10, 33, or 100 mg ferrous salt in fasted rats. Ethylene production was dose-dependent and significantly higher than control (KMB alone) for all doses of iron salt ( p < 0.01). A plateau in ethylene production was attained within the first hour for the lowest dose (10 mg). With the intermediate dose (33 mg), a burst of ethylene production occurred within the first hour. With the highest dose (100 mg), a relatively steady rate of ethylene production was seen throughout the 4-hr test period. The cumulative levels of ethylene at 4 hr, recalculated for an average rat (e.g., 250 g weight) were 7.5, 27.8,

60 J. O. Kang et aL

300

I

I

I

I

I

2

3

4

250 200 150 I00 50 0

TIME (HR) FIGURE 1. Dose-dependent production of ethylene from KMB after gastric instillation of ferrous sulfate. Fasted (18 hr) rats received sequential intragastric instillations of KMB (50 mg in 0.5 ml water) and FeSO4"7H20 (10, 33, or 100 mg in 1.5 ml) followed by a rinse with 0.5 ml water. Ascorbate was not given. Control rats received KMB (50 mg in 0.5 ml) followed by a rinse with 2.0 ml water. Data are the mean -+ SD for n = 4, except n = 3 for 33 mg ferrous salt at 3 and 4 hr. The differences between control rats and treated rats were statistically significant at all time points ( p < 0.01).

and 44.6 nmol, respectively, for injection of 10 mg (36/xmol), 33 mg (119 ~tmol), and 100 mg (360/zmol) of ferrous salt. The measured ethylene corresponds to 0.062%, 0.070%, and 0.037%, respectively, of the administered iron, based on the stoichiometry that oxidation of three ferrous ions is required (Eqs. 1-3) to give rise to one • OH. Effects o f Ascorbic Acid Formation of .OH will terminate after the supply of ferrous ions is exhausted. However, ascorbic acid can recycle the iron (Eq. 5) and, thereby, sustain the production o f • OH. On the other hand, ascorbic acid can also scavenge • OH (Eq. 7). Therefore, a biphasic action of ascorbic acid on ethylene production is expected. We compared the effects of ascorbic acid in vitro and in vivo. A fixed amount o f ferrous salt (33 mg, 119/zmol) was used with four different amounts of ascorbic acid, i.e., 0, 25, 120, and 500/zmol ascorbic acid. The same concentrations and volumes were used both in vitro and in vivo. In vitro studies were conducted in 4 mM HC1 as an approximation of gastric acidity after dilution of stomach acid [23] by the aqueous volume (2.5 ml) of administered agents. Table 2 shows the results of in vitro experiments. In the absence of ascorbic acid,

HYDROXYL RADICALS IN VIVO FROM ORAL IRON

TABLE 2. In Vitro System: Biphasic Effect of Ascorbic Acid on IMB

and Ferrous

Ethylene

Production

61

from

Salt”

Ethylene

Ascorbic acid

0.5 h

1.0 h

fimoY2.5 ml 0 25 120 500

nmollflask 256 636 332 114

f 24 (8.5) f 83 (21.2) f 21 (11.1) f 8 (3.8)

403 1313 6% 245

f f f f

1.5 h

2.0 h

(nmollflask*min)

30 (4.9) 143 (22.6) 19 (12.1) 22 (4.4)

494 1945 1046 362

f f f f

22 (3.0) 142 (21.1) 28(11.7) 34 (3.9)

561 2394 1387 465

f f f f

24 (2.2) 131 (15.0) 15 (11.4) 65 (3.4)

D Erlemneyer flasks contained the following constituents in 2.5 ml of distilled water: 50 mg KMB (294 pmol, 118 mM), L-ascorbic acid (as indicated in the table = 10,48, and 200 mM), and 33 mg ferrous salt (119 pmol, 48 rnhf). Flasks were shaken at 37’C and aliquots of gas phase (0.5 ml) were removed by syringe and analyzed for ethylene. The cumulative amount of ethylene and the rate for each 30-min interval are shown. Data are the mean f SD for five separate experiments.

the initial rate of ethylene production (8.5 ntnol/min) diminished in successive OS-hr

time intervals. The final recorded rate (2.2 nmol/min), was only 26% of the initial rate. When 25 pm01 of ascorbic acid were added (10 mM final concentration), the initial rate was increased by 148 %, and the rate was maintained through 1.5 hr; the rate of ethylene production then fell by 29% in the fourth time interval. The total ethylene production at 2 hr in the presence of 25 pm01 ascorbic acid was 327 % greater than that seen in the absence of ascorbic acid. When the ascorbic acid was increased to 120 pm01 (48 mM), the initial rate of ethylene production fell back to only 30% above control (no ascorbic acid); this rate was maintained through 2 hr. The total ethylene at 2 hr was 147% greater than the control. When 500 rmol of ascorbic acid were added (200 n&l), the initial rate was suppressed to only 45% of control and remained relatively constant throughout the 2-hr experimental period. The total ethylene produced was 83 % of control. Thus, a clear biphasic action was seen in which lower concentrations of ascorbic acid increased the initial rate of ethylene production, but higher concentrations suppressed the rate. However, ascorbic acid also tended to maintain a constant rate of ethylene production in vitro, so that the amounts of ethylene, with and without 500 pm01 ascorbic acid, differed by only 17% at the end of 2 hr. During the course of the study, it was noted that a faint orange-to-pink color developed upon mixing the solutions of KMB and ferrous salt. A purple hue intensilied during the incubation at 37°C. The presence of ascorbic acid suppressed purple color development. The same purple color was formed instantaneously on addition of ferric sulfate solution to the reaction mixture, but ascorbic acid blocked color formation. The color formation during incubation or upon direct addition of ferric salt, and the suppression of color by ascorbic acid, are best attributed to the formation of a complex or chelate between ferric ions and KMB, and to the reduction of ferric iron to the ferrous state by ascorbic acid. Similar experiments were conducted in vivo by gastric instillation of the components (Table 3). The same total volume and amounts of KMB, ascorbic acid, and ferrous salt were used as for the in vitro studies. The rate of production of ethylene

62

J. 0. Kang et al.

in the absence of ascorbic acid was highest initially and fell off thereafter. The ethylene detected in vivo was much less than that seen in vitro: The initial rate (recalculated from Table 3 for an average 2.50-g rat) was 0.25 nmolkatlmin, which represented 3.7% of that seen over the same time span in vitro (403 nmol at 1 hr, or 6.7 nmollmin, Table 2). Similarly, the total ethylene at 2 hr (20.5 nmol/rat) was 3.7% of that seen at 2 hr in vitro (561 nmol, Table 2). When 25 pmol of ascorbic acid were co-injected, neither the initial rate of ethylene production, nor the subsequent decline in rate, were affected. The initial rate and the cumulative ethylene at 2 hr (per 250-g rat) were 1.2 % and 0.8%, respectively, of the corresponding in vitro values. When the amount of ascorbic acid was raised to 120 Fmol, a modest increase in initial rate was seen (Table 3), but it did not achieve statistical significance (p > 0.2). The subsequent time intervals were not significantly different from control (iron alone). The initial rate and the cumulative ethylene at 2 hr were 2.9% and 1.9%, respectively, of the corresponding in vitro values. When 500 pmol of ascorbic acid were coinjected, the cumulative ethylene compared to control was 44% (p < O.Ol), 48% (p < O.Ol), 57% (p < 0.05), and 61% (p > 0.1) at 1,2, 3, and 4 hr, respectively. The initial rate and the cumulative ethylene at 2 hr were 2.7 % and 2.1% , respectively, of the corresponding in vitro values. Effects of .OH Scavengers Scavengers of . OH can suppress the yield of ethylene by competing with KMB for the evanescent .OH. Two potent scavengers, MqSO and ethanol, were tested (Fig. 2). The second order rate constants (kj for reaction with *OH are: Me$O = 7.1 x lo9 M-l s-l and ethanol = 1.7 x lo9 M-’ S-I [24]. Thus, the reaction of these agents with -OH can be considered to be essentially diffusion limited. Urea, a very poor scavenger (k < 7 x lo5 Mm’ s-’ [24]), was used as a negative control. Results with ascorbic acid (k = 1.2 x 1O’OM-l s-l [24]) were presented earlier in Table 2. Figure 2 shows that MeSO and ethanol each suppressed ethylene production in water or in 4 mM HCl. The control rate of ethylene production was less in 4 mM HCl compared to water (9.2 f 0.6 versus 15.5 k 1.4;~ < 0.01). Suppression of ethylene by the scavengers was dose-dependent and, in general, MQSO was more effective

TABLE 3. Rats in Vivo: Effect of Ascorbic Acid on Ethylene Production from Intragastric KMB and Ferrous Salt’ Ethylene Ih

Ascorbic acid pm01 0 2s 120 500

3h

2h nmollkg

59 61 82 26

* * * *

11 14 53 11

(0.98) (1.02) (1.37) (0.43)

82 80 106 39

f f * *

24 22 57 18

(0.38) (0.32) (0.40) (0.22)

4h

(nmollkg ‘min) 95 93 113 54

f f f *

36 29 61 25

(0.22) (0.22) (0.12) (0.25)

99 95 119 60

* * zt *

44 27 65 31

(0.07) (0.03) (0.10) (0.10)

O Fasted (18 hr) ~at.sreceived sequential gastric instillations of KMB (50 mg. 294 @nol) in 0.5 ml water, a fresh mixture of L-ascorbic acid (as indicated in the Table) and ferrous salt (33 mg, 119 @toI) in 1.5 ml water, and 0.5 ml water as a rinse. The cumulative amount of ethylene and the rate during each 60min interval are shown. Values are the mean *SD for n = 6.

HYDROXYL RADICALS IN VIVO FROM ORAL IRON

0

0

0.08

0.4

SCAVENGER

0.8 CONCENTRATION

0

63

0.8

(Ml

FIGURE 2. Effect of *OH scavengers on the formation of ethylene from KMB and ferrous salt in vitro. Capped Erlenmeyer flasks at 37°C contained KMB (14.4 mM), *OH-scavengers at the concentrationsindicatedin the figure, and ferrous salt (2.8 mM), in a final volume of 2.5 ml in water or in 4 mM HCl. Reactions were initiated by addition of the solution of the ferrous salt. Values shown are the mean f SD, n = 3. Numbers in parentheses are the percent of control.

than ethanol, in agreement with their relative rate constants. Urea did not exhibit an inhibitory action, in accord with its comparatively poor rate constant. However, increases in yield of ethylene were observed in the presence of 0.8 M urea (Fig. 2); the increases may be related, in part, to slight increases in pH due to the base strength of urea (pH 3.25 versus 3.57 in 4 mM HCl, without and with 0.8 M urea, respectively, and, correspondingly, pH 5.88 versus 6.18 in water). The comparative effects of the scavengers (MeSO > ethanol >>> urea) are in general accord with comparative effects in other systems, such as ethylene production by activated liver microsomes [25] or ring-hydroxylation of dopamine by iron/EDTA/ascorbate [26]. Effect of Catalase If ethylene production from the interaction of ferrous salt and KMB is the result of -OH formation from the sequential reactions described in Eqs. 1-3, then interruption of the sequence should diminish the production of ethylene. We investigated the effect of catalase, an enzyme that catalyzes the dismutation of Hz02 (lQ. 9): 2H202 + O2 + 2H20

(9)

Figure 3 shows that catalase markedly suppressed ethylene production. Heatinactivated catalase was comparable to an identical concentration of bovine albumin, which was used as a protein control. These data indicate that Hz02 is a required intermediate for the formation of ethylene. Hence, a Fenton reaction between ferrous ions and generated HZ@ (Eq. 3) is indicated. Some production of ethylene was noted in control samples where the ferrous salt was omitted (Fig. 3; bovine serum albumin, no Fe* +) . Added ferrous salt plus catalase gave a lesser yield of ethylene (Fig. 3). These results indicate probable trace contamination of the reagents (ascorbate, KMB, and/or serum albumin) with iron. In

64 J. 0. Kang et al.

35-

I

I

I

I

T

A

Fe2++ Bovine serum albumin-

30/

325-

_ A..‘)<

i

te’L+

Inactivated catalase

50 520Y ti l5z L IO5

x/U

Bovine serum albumin 1 (no Fe*+) (control)

-

_ _

Fe’+ +

0

a-T-~Catalase

4

8

12 16 20 TIME (MINI

FIGURE 3. Effect of cat&se on the formation of ethylene from KMB and ferrous salt in vitro. Experiments were conducted in distilled water rather than 4 mM HCl, and with more dilute reactants, in order to avoid inactivation of the cat&se. Capped Erlenmeyer flasks at 37°C contained KMB (2.4 mM), ascorbic acid (0.20 mM), and ferrous sulfate (0.48 mM) in a final volume of 2.5 ml. Catalase protein or bovine serum albumin (as control) was present at 0.7 mg per 2.5 ml. Inactivation of cat&se was achieved at 100°C for 15 min. Reactions were initiated by addition of the solution of ferrous salt. Values shown are tbe mean + SD, n = 5, except n = 3 when iron was omitted.

separate experiments, DTPA, an iron-chelating agent, was added to the reaction mixture. The ferric-DTPA chelate is not readily reduced by either superoxide or ascorbate; hence, trace iron is effectively removed Corn participation in the reaction sequence that leads to -OH. The yield of ethylene was suppressed by 50 PM DTPA even with the addition of 1 mM H20z (0.4 f 0.1 nmol ethylene at 20 min) compared to the control in Figure 3. These results confirm trace contamination of the reagents with iron and show that HzOz alone (no ferrous ions) does not yield ethylene from KMB. DISCUSSION Although hydroxyl radicals are often implicated in cell injury, direct assessment of their involvement is impeded because they are too reactive to accumulate in tissues in measurable concentrations. KMB can be useful in circumventing this obstacle. It is a

HYDROXYL RADICALS IN VIVO FROM ORAL IRON

65

potent scavenger that produces a gaseous product, ethylene, which is readily measured by gas chromatography. Ethylene can be exhaled [2 l] ; in the current study, it may have been partially expelled from the stomach into the chamber air. An advantage to utilizing KMB is that the product, ethylene, accumulates in the enclosed chamber atmosphere and, therefore, can be followed over time for individual rats. To our knowledge, the experiments with oral iron represent the first successful application of this technology to study *OH in vivo. KMB (a keto-acid) and its aldehyde analogue, methional, have been used in numerous in vitro studies to detect -OH [e.g., 19, 20, 25, 27-291. Alkoxyl radicals also give rise to ethylene [30]. KMB had been used in vivo previously [31] in an attempt to detect alkoxyl radicals that arise during lipid peroxidation. Although rats produced ethylene from KMB after i.p. injection of carbon tetrachloride, an agent that induces hepatic lipid peroxidation, the source did not appear to be alkoxyl radicals because alpha-tocopherol, a lipid antioxidant, failed to suppress ethylene production, although it did suppress lipid peroxidation. In the current experiments, the inhibition of ethylene production by catalase (Fig. 3) shows that H202 is a required reaction intermediate (cf. Eq. 3). The inhibition of ethylene production by *OH scavengers (Fig, 2), in concordance with their relative rates of reaction with *OH, provides confirmatory evidence that -OH is responsible for the generation of ethylene from KMB. The present study shows that the in vivo production of *OH is dose-dependent in the range of 10-100 mg FeS04(7H20) administered per 220-250-g rat (Fig. 1). In toxicological studies with rats [7], a dose of 320 mg elemental iron/kg, equivalent to 400 mg FeS04(7H20) per 250-g rat, was lethal. Thus, our studies were conducted well below the lethal range. In prior studies [7], damage to tissues of the stomach became evident at doses of 80 mg elemental iron/kg (100 mg ferrous salt/rat). The detection of substantial amounts of ethylene (Fig. 1) after administration of only 10 mg ferrous salt/rat (8 mg elemental iron/kg) suggests that significant damage by -OH may occur at a molecular level before tissue damage becomes evident histologically. In Figure 1, ethylene production for a low dose of 10 mg ferrous salt reached a plateau at 1 hr. At the highest dose of 100 mg oral iron, vigorous production of ethylene was observed for up to 4 hr. In the presence of ascorbate (Table l), production of ethylene persisted through 7 hr. The early formation of ethylene probably reflects *OH production within the stomach cavity. However, it is improbable that sufficient amounts of KMB remained in the gastrointestinal tract throughout the extended time course. Therefore, ethylene production at later times may reflect formation of ethylene in blood and/or internal organs. After KMB has been absorbed and redistributed, its concentration is decreased, and, moreover, it must compete with tissue ascorbate and glutathione in concentrations as high as 10 mM, as well as with the sum total of all other tissue compounds. On the other hand, after an overload of oral iron, an increase in concentration of low molecular weight iron chelates or complexes (or protein bound iron) becomes probable. Autoxidation of low molecular weight ferrous chelates becomes exceptionally rapid at neutral pH [32]; in combination with recycling of iron by tissue reducing agents, the more rapid autoxidation could facilitate ethylene production in vivo. These conflicting factors deserve more detailed exploration. Ascorbic acid can, in theory, amplify -OH production by recycling iron from the oxidized to the reduced state [ 15,331. Amplified production of ethylene was observed in vitro when 25 pmol of ascorbic acid were added to 119 gmol ferrous salt in solution

66

J. 0. Kang et al.

(Table 2). However, the increased production of ethylene was greater than expected. Since 25 pmol of ascorbic acid can reduce 50 pmol of ferric ion (Eq. 5), an increase by as much as 42% in the initial rate can be anticipated. The observed increase was 148 % . We have no direct explanation for this phenomenon. Perhaps, the ferric-KMB complex (purple color, see Results) does not yield ethylene: however, the relative amounts (Table 2, 294 pmol KMB and 119 pmol ferrous salt) make it unlikely that reduction of a ferric-KMB complex by ascorbic acid could triple the rate of ethylene production. Another possibility is that iron aggregates that form in concentrated solution [34] may complex some Hz02; in that event, reduction of Fe3 + to Fez+ may free bound Hz02 to participate in the Fenton reaction (Eq. 3)., Ascorbic acid can also suppress the yield of ethylene by competing with KMB for reaction with -OH. A distinct suppression of yield of ethylene was observed both in vitro (Table 2) and in vivo (Table 3) when 500 pmol of ascorbic acid was mixed with 119 pmol ferrous salt. Thus, high concentrations of ascorbic acid may exert an overall protective role. Table 4 provides a kinetic analysis of the expected reaction rate of -OH with each component of the in vitro experiments shown in Table 2. Methionine served to approximate the rate constant for KMB. The values of the rate constant multiplied by the molar concentration are consistent with the experimental results shown in Table 2. At the lowest concentration (10 mM), ascorbic acid was not an effective competitor with KMB (Table 4), and increased production of ethylene was seen (Table 2). The middle concentration of ascorbic acid (48 mM) was competitive with KMB, and stimulation of ethylene was lessened. At the highest concentration (200 mM), ascorbic acid was a very effective competitor, corresponding to the observed marked suppression of ethylene both in vitro (Table 2) and in vivo (Table 3). The high concentration of KMB used in vitro (Table 2) deserves comment. Most experiments in the literature used concentrations in the range of 10 mM, with very much lower concentrations of *OH-generating agent. In Table 2, we used 118 mM. A high concentration was necessitated by the concentration of ferrous salt (48 mM), which, in turn, was based on the toxicological literature for amounts producing tissue damage in rats. KMB was used in excess in order to facilitate scavenging of *OH in

TABLE 4. Bimolecular Rate Constants for the Reaction of *OH with the Components of the In Vitro Experiments,

and Product of the Rate Constant

Component

k(*OH) (M-’ s-‘)

Fez+ KHso4 KMB (Methionine) Ascorbic acid

5.0 1.5 6.0 1.2

x x x x

108 106 lo9 10’0

Concentration Used (M) 0.048 0.048 0.118 0.010 0.048 0.200

x the Concentration” k(.OH)

x Concen. (s-9

2.4 7.1 7.1 1.2 5.8 2.4

x x x x x x

10’ 104 108 108 lo* lo9

’ Rate constants are from Dorfman and Adams [24]. The rate constant for KMB is not available; methionine, the corresponding amino acid, was used as a reasonable approximation for KMB. The products of the rate constants x the concentrations provide estimates of the relative rates of reaction of .OH with each of the components of the reaction in Table 2.

HYDROXYL

RADICALS

IN VIVO FROM ORAL IRON

67

competition with scavenging directly by ferrous ions (Table 4). The scavenging action of ferrous ions is implicit in the overall stoichiometry for the oxidation of Fez+ by Hz02 (Eq. lo), which is comprised of a Fenton reaction (Eq. 3) followed by Eq. 11. Sulfate was not an effective competitive scavenger (Table 4). 2FeZ+ +H202+2H++2Fe3+ Fe2+ + H+ + *OH+Fe3+

+2H20 + H20

(10) (11)

The yield of ethylene produced in vivo in the absence of ascorbic acid was only 3.7 % of the corresponding yields seen in experiments conducted in vitro with the same amounts and concentrations of reactants (Tables 2 and 3). Hydrocarbon gases can be metabolized by rodent tissues [35-381. When ethylene (7-124 nmol) is injected i.p. into mice, it is exhaled over the course of 30 min or less with a mean recovery of 34% [21]. However, this does not account for the much lower yield seen in the current experiments. The lower yield may be due to a number of factors: 1) decreased availability of oxygen to support autoxidation reactions in the stomach cavity, 2) loss of generated -OH by reaction with biomolecules in the stomach, and 3) lesser recovery of ethylene formed in the stomach. When 25 pmol of ascorbic acid were added, the in vivo yield of ethylene fell from 3.7% to 0.8-l .2% of that seen in vitro, reflecting the stimulation of ethylene production in vitro, but not in vivo. With higher amounts of added ascorbate (120 hmol and 500 pmol), where scavenging of -OH becomes a factor, the in vivo yield was in the range 2.7-2.9 % (1 hr) and 1.9-2.1% (2 hr) of that seen in vitro. At low pH, the production of *OH during the reaction of ferrous ions with H202 is well accepted 1241. However, recent studies at or near neutral pH indicate that additional intermediates may be generated during Fenton-type reactions between H202 and certain ferrous chelates [39, 401 or enzymatically bound iron [41]. Indirect evidence indicates possible formation of higher oxidation states of iron [42] or ferrous peroxide complexes. Although some of the postulated intermediates are strong oxidants (e.g., the ferry1 ion [Fe02+] [43]), the reactivity may differ from *OH. For example, an intermediate produced by xanthine oxidase [41] is not effectively scavenged by Me2S0. In our study, however, MezSO was effective in suppressing ethylene production in vitro during the autoxidation of ferrous ions at low pH (Fig. 2). Nonetheless, the presence of a variety of endogenous ligands in vivo may result in the formation of higher oxidation states of iron, which could contribute to tissue damage. It is also recognized that the presence of oxygen can result in complex reactions with tissue constituents in which alkoxyl radicals, peroxyl radicals, and other radical intermediates are generated; these intermediates can also contribute to tissue damage. A number of suggestions have been made about factors that play a role in iron poisoning. Spencer [44] postulated that death from iron poisoning may be the result of shock produced from hemorrhagic necrosis and fluid loss from the gastrointestinal tract. Latner, quoted by Spencer [44], attributed the toxicity of oral iron to its oxidizing capacity; iron may inactivate biomolecules that contain sulthydryl groups, thereby altering cellular metabolic processes. Brown and Gray [45] proposed that circulating unbound iron acts as a vasodepressant, thereby creating vascular shock. It is known that oral iron enters the blood stream and tissues, and that deposition of granular iron in the ferric form can be seen by histochemical stain [2]. Nonetheless, the molecular mechanism(s) responsible for the toxicity of iron are unknown. We suggest that oral iron generates *OH in the stomach and proximal small bowel.

68

J. 0. Kang et al.

An overload of oral iron may result in formation of -OH in body fluids and tissues. Hydroxyl radicals are extremely damaging and react avidly with molecules present at their site of generation. Carbohydrates, such as glucose and ribose, are highly reactive [24]. The stomach is covered with mucin, which has a high content of polysaccharide [46]. A destructive action on the mucous surface of the stomach can be expected. Hydroxyl radicals produced on homolytic scission of water are responsible for much of the tissue damage seen after exposure to high-energy radiation [47,48]. Therefore, hydroxyl radicals (and/or higher oxidation states of iron) may mediate tissue damage and general toxicity seen after accidental poisoning with oral iron preparations. Adam Slivka was supported by Medical Scientist Training Grant GM-07280.

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Received July 22, 1988; accepted July 26, 1988