Effect of the antihypertensive drug hydralazine on mineral metabolism in the rat

Toxicology Letters,

41 (1988) 193-202

193

Elsevier

TXL 01958

Effect of the antihypertensive drug hydralazine mineral metabolism in the rat

Jeffrey

M. Peters’,

Bo Liinnerdallp2

on

and Carl L. Keen132

Departments of ‘Nutrition and ‘Internal Medicine, University of California, Davis, CA 95616, U.S.A. (Received

23 December

(Accepted

20 January

1987) 1988)

Key words: Manganese;

Copper;

Iron;

Drug-nutrient

interaction;

Hydralazine;

(Rat)

SUMMARY The effects of hydralazine were fed HZ (O.l-4%, 2-month

long-term

removed

and concentrations

were similar.

(HZ) on mineral

w/w)

in three

metabolism

separate

was investigated

experiments:

feeding study; and (3) a pregnancy

(1) a 2-week

HZ had no effect on tissue Mn and Zn concentrations,

of HZ on mineral

metabolism

were higher in HZ-treated

may be, in part,

dose-response

study. At the end of each experiment,

of Mn, Cu, Zn and Fe were measured.

lower and kidney Cu concentrations

in rats. Sprague-Dawley

responsible

groups

study;

rats (2) a

tissues were

Results from the three experiments while tissue Fe concentrations compared

to controls.

for some of its negative

were

The effect

side effects.

INTRODUCTION

Hydralazine (HZ) (1-hydrazinophthalazine) has been used as an antihypertensive drug for over 30 years [l]. The usefulness of this drug, however, is compromised by severe side effects including lupus-like complications which can develop in subjects receiving the drug for a prolonged period [3-51. These side effects are usually reversible and do not result in permanent damage if HZ therapy is discontinued [3]. In 1960, Comens [2] reported that HZ-induced perosis in small chicks was prevented by supplemental manganese (Mn) given orally. Based on this observation, Comens suggested that HZ induced a Mn deficiency in the animals which was in part responsible for the drug-associated pathologies. This investigator also reported that

Address

for correspondence:

ty of California, Abbreviation:

037%4274/88/$

Davis,

Carl L. Keen, Departments

of Nutrition

and Internal

Medicine,

CA 95616, U.S.A.

HZ, hydralazine.

03.50 0

1988 Elsevier

Science Publishers

B.V. (Biomedical

Division)

Universi-

194

dogs which had received oral HZ developed renal lesions and had circulating lupus cells, which also could be prevented by supplemental Mn. Further evidence of a potential relationship between HZ and Mn was suggested in 1963 by Hurley et al. [6] who reported that prior injection of Mn salts had a protective effect against HZinduced seizures in rats. While the above reports suggest that an interaction between HZ and Mn may occur, it is important to note that tissue Mn levels were not measured in the above studies. Recently, it has been pointed out that the relationship between HZ and Mn is still unclear [7]; a fact which is disturbing given the widespread use of this drug in clinical practice. In the present study, we investigated the effects of HZ on Mn metabolism in a rat model; in addition we investigated the effect of the drug on zinc (Zn), iron (Fe) and copper (Cu) metabolism to ascertain if any observed effects of HZ on Mn metabolism were specific for that element, or were reflective of a general effect of the drug on mineral metabolism. MATERIALS

AND

METHODS

Experiment 1: Dose-response Virgin female Sprague-Dawley rats (180-200 g) were purchased from a commerGilroy, CA) and individually housed in cial vendor (Simonsen Labolratories, stainless steel cages in a light- and temperature-controlled room (12 h-12 h; 23°C). After one week of acclimation, rats were divided into four groups and received one of the following diets: 1 pg Mn/g diet (D), D + 0.1% HZ, D + 0.2% HZ, or D + 0.4% HZ. The 1 pg Mn/g diet represents a Mn-deficient diet [8]. This diet was used in the initial experiment to maximize the potential of identifying an effect of HZ on Mn metabolism. The detailed composition of the diet has been described previously [8]. Food intake was recorded daily and body weights were measured every 3 days. After consuming their respective diets for 14 days, rats were anesthetized by overexposure to carbon dioxide and blood was removed by cardiac puncture. Hematocrits were measured for all blood samples. Liver, kidney, intestine, heart and brain were quickly removed, weighed and frozen in snap-cap vials. Tissues were wet-ashed using 12 N nitric acid [9], and analyzed for Mn, Zn, Cu and Fe concentrations using flame atomic absorption spectrophotometry (Instrumentation Laboratories Model 551, Wilmington, MA). Recovery of these minerals by this method is between 98 and 102% [9]. Values are expressed as pg element/g tissue wet weight. Experiment 2: Long-term administration Virgin female Sprague-Dawley rats (180-200 g) were housed as described After a 7-day acclimation period, rats were divided into groups and were of the following diets: 1 pg Mn/g diet (D), D + 0.2% HZ, 5 pg Mn/g diet + 0.2% HZ, 45 pg Mn/g diet (C), or C + 0.2% HZ. The 5 pg Mn/g diet

above. fed one (M), M and the

195

45 pg Mn/g

diet represent

were recorded

marginal

and control

Mn diets, respectively.

every 3 days as were body weights.

After

receiving

Food intakes their respective

diets for 2 months, rats were anesthetized by overexposure to carbon dioxide and blood was removed by cardiac puncture. Hematocrit was measured in all blood samples. Liver, kidney, intestine, brain and heart were removed, weighed and frozen. Tissues were wet-ashed and analyzed for Mn, Cu, Zn and Fe concentrations as described above.

Experiment 3: Administration

during pregnancy

Virgin female Sprague-Dawley rats (180-200 g) were housed as described above. After a 7-day acclimation period, rats were divided into four groups and were fed one of the following diets: M diet, M + 0.2% HZ, C diet, or C diet + 0.2% HZ. Food intakes and body weights were recorded as described above. After consuming their respective diets for 14 days, rats were mated overnight with male SpragueDawley rats. Presence of sperm plugs and/or positive vaginal smear was indicative of pregnancy and considered day 0 of gestation. On day 21 of gestation, pregnant rats were overexposed to carbon dioxide and blood was removed by cardiac puncture. Hematocrit was measured in all blood samples. Maternal liver, kidney, heart, brain and intestine, and placentas and fetuses were removed. Tissues and fetuses were weighed, wet-ashed and analyzed for Mn, Cu, Zn and Fe concentrations as described above. Fetuses were dried at 100°C for 48 h prior to ashing, and percent dry weight was calculated to determine if HZ resulted in fetal edema. While it can be argued that injecting HZ represents an alternative method to approach the present investigation, we chose to feed the drug in the current study since HZ is typically administered orally to hypertensive patients [ 10,111.

Statistics Data from the dose-response and 21-day pregnancy experiments were analyzed by one-way analysis of variance with Duncan’s multiple-range test being used to determine statistical differences between groups. Data from the long-term experiment were analyzed by two-way analysis of variance and Scheffe’s test was used to determine statistical differences among groups [ 121. RESULTS

Dose-response experiment Compared to dams not fed the drug in their diet (Fig. 1). first week. HZ had no consistent effect Cu concentrations in groups drug; however, this difference

HZ, food intake was depressed in all groups receiving This difference in food intake was not noted after the on tissue Zn or Mn concentrations (Table I). Kidney fed HZ were higher compared to dams not fed the was not statistically significant (Table I). Liver and

196

kidney Fe concentrations were significantly lower in groups receiving HZ compared to controls (Table I). Plasma Cu, and Fe concentrations were not consistently affected by HZ feeding. No differences in hematocrit levels or tissue weights were observed among the groups. Long-term experiment Similar to the results in the dose-response experiment, food intake was initially depressed in HZ-treated groups relative to controls. However, no difference in food intake was observed after the first week (Fig. 2). Dietary Mn had no effect on food

0

3

6

9

12

15

Day Fig. 1. Food intake graph for the dose-dependent experiment. Dietary groups: Mn deficient (1 pg M&g diet) ( q ), Mn deficient + 0.1% HZ ( A ), Mn deficient + 0.2% HZ ( n ), Mn deficient + 0.4% HZ ( A ). Each point represents

the mean

from four animals

* SEM.

* Significant

difference

at P~0.05.

197

Dietary Mn had a significant effect on liver and kidney Mn concentrations. Liver Mn levels were lower in D and M groups than in controls, while kidney Mn levels were lower in D rats than in M and C rats (Table II). HZ did not significantly affect tissue Mn levels in any of the groups, although there was a trend of higher liver Mn concentrations in rats receiving HZ. Neither dietary Mn nor HZ had a consistent effect on tissue Zn concentrations. Similar to the dose-response experiment, liver and kidney Fe concentrations were lower in some of the groups receiving dietary HZ compared to controls, while kidney Cu was significantly higher in rats fed HZ compared to their respective controls (Table II). No differences in hematocrit or tissue weights were observed among the groups. Administration during pregnancy experiment Similar to the above experiments, the initial food intake depression in groups receiving HZ in their diet was overcome after one week. Food intake during pregnancy did not differ among groups. Dietary Mn had a significant effect on tissue Mn concentrations; dams in groups receiving the marginal Mn diet had lower

TABLE TISSUE AZINE

I Mn, Cu, Zn AND Fe CONCENTRATIONS

IN CONTROLS

AND

RATS FED HYDRAL-

(HZ) FOR 2 WEEKS

Concentrations

are expressed

as gg/g

Mn

Dietary

tissue wet weight;

values are means Zn

cu

k SEM. Fe

n

4

group Liver 1.3

k 0.1a.b

4.5 * O.lasb

26.7

+ 0.2”

331 * 22b

D+O.l%

HZ

1.1

* 0.1=

4.7 + O.lb

27.8

+ 0.3=

246 + 4a

4

D+0.2%

HZ

1.0

f

4.1 f

27.7

+ l.7a

270 + 12a

4

D+0.4%

HZ

1.5

+ O.la

3.6 + 0.4”

28.6

+ 0.9”

268 + 13=

4

0.56

k O.Ola*b

5.6 + O.l=

25.5

+ 0.5a

87 k 5b

4

D+O.l%

HZ

0.44

+ 0.05”

8.0 k 0.2a

25.6 f

0.4”

72 k 5”

4

D+0.2%

HZ

0.53

k 0.06asb

8.3 k 1.8=

27.0 f

0.7a

77 f

3a.b

4

D + 0.4%

HZ

0.62

+ 0.06b

26.5

0.8”

68 f

2a

4

D

0.1=

0.4a.b

Kidney D

Plasma D

10.8 f

4.3a

f

_*

1.4 + O.la

D+O.l%

HZ

_*

1.4 + 0.3”

1.07 f 0.93 f

D+0.2%

HZ

_*

1.5 + 0.2a

0.82

D+0.4%

HZ

_*

1.2 + 0.7a

D = Mn-deficient

diet (1 pg Mn/g

*Below detectable

concentration.

“sbValues within Duncan’s

a column

multiple-range

0.06b 0.03a.b

5.8 + l.3a 3.2 + 0.4a

4 4

+ 0.05”

3.5 * 0.3=

4

1.07 rt 0.07a

4.5 * l.3a

4

diet).

with the same superscript test, P
letter represent

homogenous

subgroups,

based on

198

Mn concentrations

in liver and kidney

than did controls

(Table

III). Fetal Mn con-

centration was not affected by dietary Mn level. HZ feeding had no significant effect on Mn concentrations in any of the maternal tissues analyzed, although again there was a trend of higher liver Mn in HZ animals fed the M diets compared to their respective controls. Fetal Mn concentrations were not affected by HZ on either a wet or dry basis. Maternal and fetal tissue Zn concentrations were not significantly different among groups. Although not statistically significant, maternal liver,

6

0 0

6

12

18

24

30

36

42

48

54

60

Day Fig. 2. Food intake graph

for rats fed HZ for 2 months.

(0, Mn deficient

HZ (O), marginal

control

+ 0.2%

(45 kg M&g

Dietary

Mn (5 pg Mn/g

groups:

Mn deficient

diet) ( q), marginal

diet) ( A ), control + 0.2% HZ ( A ). Each point represents three animals. * Significant difference at PC 0.05.

(1 pg Mn/g

Mn + 0.2%

diet

HZ (w),

the mean from at least

199

TABLE

II

TISSUE AZINE

Mn, Cu, Zn AND Fe CONCENTRATIONS

IN CONTROLS

AND RATS

FED HYDRAL-

(HZ) FOR 2 MONTHS

Concentrations

are expressed

as pg/g

cu

Mn

Dietary

tissue wet weight;

values

are means

Zn

k SEM. Fe

n

group Liver D

0.06” 1.03 * 0.05b

5.2 k 0.6a

22.2

f 0.9a

385 k 29”

4

4.6 k 0.1”

22.6

k l.la

3

0.63

5.8 f

23.1

f

356 + 20a 348 f 63” 155 * 34b 219 zk 24b 251 f 45b

3

0.66 f

D+0.2%

HZ

M M+0.2%

HZ

C C+O.2%

f

0.20b

l.ga

1.09 + 0.17b 1.63 k 0.16a

5.5 k 2.4a 7.9 Xk 1.9a

HZ

1.77 * 0.20=

4.8 f

HZ

0.44

+ 0.04b f 0.05b

0.52

k O.lla

0.54

l.Oa

31.9 + 6.8b 25.0 f l.Oa 24.8 + 0.5=

0.5=

3 8 9

Kidney D

0.24

D+0.2% M M + 0.2%

HZ

L

0.59

k O.loa f 0.03a

C+O.2% HZ

0.62

+ 0.07”

8.45 + 0.42a 18.1 t l.gb

23.4

+ 0.9a

27.7

-+ 1.4a

0.5a

23.4

+ 3.0a

* 4.4b

23.9

+ 2.0a

8.4 + 0.5a 17.8 k 3.2b

22.8

k l.7a

25.6

k l.2a

0.82

+ 0.09” 1.05 + 0.03a*b

0.68

+ 0.03”

0.64

k 0.03a

0.8 + O.la 1.12 f 0.01a.b 1.20 + 0.08asb

1.08 k 0.08” 1.03 k 0.02a 1.02 + O.lla

1.33 t

1.08 + 0.21=

7.8 f 22.1

131 + lla 111 + 8”

4

96 + 12= 83 t 15a 104 * 1oa

3

95 f

7a

3 3 8 9

Plasma D D+0.2%

HZ

M M + 0.2%

HZ

C C+O.2%

HZ

D = Mn-deficient pg Mn/g

_* _* _* _* _* _* diet (1 gg Mn/g

0.07b

diet); M = marginal

4.5 f 0.8” 2.9 + O.l=

4

3.9 k 0.7a 3.7 * O.la

3

3.6 f 0.4a 4.1 + 0.6a

Mn diet (5 pg Mn/g

3 3 8 9

diet); C = control

diet (45

diet).

*Below detectable a*bValues within Scheffe’s

concentration. a column

with the same superscript

letter represent

homogenous

subgroups,

based on

test, PcO.05.

kidney and plasma Fe concentrations were lower in HZ-fed groups compared to controls. Maternal kidney Cu concentrations were significantly higher in all groups receiving HZ in their diet compared to controls (Table 111). Fetal Fe and Cu concentrations were not affected by HZ (Table III). No differences in hematocrit or tissue weights were observed among groups. DISCUSSION

Reports from Comens [2] and Hurley et al. [6] suggested that some of the side effects associated with HZ were due in part to a drug-induced Mn deficiency.

200

TABLE

III

MATERNAL

AND

FETAL

TISSUE

AND RATS FED HYDRALAZINE Concentrations

are expressed

as Ic.g/g tissue wet weight;

Mn

Dietary

Mn, Cu, Zn AND

Fe CONCENTRATIONS

(HZ) THROUGHOUT

values are means Zn

cu

IN CONTROLS

PREGNANCY + SEM. Fe

n

group Maternal

liver

M M + 0.2%

HZ

C C+O.2%

HZ

Maternal

kidney

M M + 0.2%

HZ

C C+O.2%

HZ

Maternal

plasma

M M + 0.2%

HZ

C C+O.2%

HZ

0.94

t

0.06a

4.0 + 0.3a

23.7

k 0.5a

163 f 46a

5

1.6

f

0.3”

24.2

+ 1.4a

1.8

+ 0.1”

3.6 * 0.3” 7.0 + 4.8”

24.0

f

119 + 18a 132 + 21a

5 3

1.3

f 0.2”

2.4 k 0.3a

23.2

k l.Oa

110 + 6a

3

0.46

+ 0.05a

4.9 k 0.4a

22.5

t

0.58

+ 0.02”

17.0 + 3.3b

0.75

* 0.14a

8.1 + 4.3”

0.84

+ 0.08b

20.5

f

3.6b

0.8”

1.9a

lla

5

25.3 + 2.2a

93 t

72 + 8=

5

24.3

-+ 2.1a

98 + 13a

3

22.5

k l.la

79 + 2=

3

1.21 k 0.08=

3.8 + 0.5”

5

1.19 t

3.8 + 0.5=

5

_*

1.12 + O.Osa

-* _*

1.2 * 0.2” 0.8 k 0.4a

0.4 + 0.2a

5.4 t

2.2=

3

-*

1.4 * 0.1”

1.1 t

2.6 f

0.8”

3

0.09= O.la

Fetuses M M+0.2%

HZ

C C+O.2%

HZ

M = marginal

Duncan’s

f

0.02a

1.45 * 0.03a

18.3 + 1.3a

56 + 3a

5

0.13

f

0.03”

1.6

f

19.2 t

0.9”

60 + 3a

5

0.17

f

O.Ola

1.2

+ 0.2=

19.0 + 0.8a

60 + 3=

3

0.29

f

O.Olb

1.68 f 0.08=

18.6 + 0.9=

56 + 4=

3

Mn diet (5 pg Mn/g

*Below detectable “.bValues within

0.14

O.la

diet); C = control

diet (45 gg Mn/g

diet).

concentration. a column

multiple-range

with the same superscript

letter represent

homogenous

subgroups,

based on

test, PC 0.05.

However, results from the present investigation show that tissue Mn concentrations were unaffected by HZ administration under varying conditions. The dose-response experiment indicated that after a relatively short period (14 days) of HZ administration, no significant alterations in tissue Mn levels occurred. Long-term HZ administration (2 months) as well produced no significant changes in tissue Mn levels although a tendency towards increased liver Mn in HZ animals was noted. The pregnant rat model was used to investigate if an additional stress on body Mn pools would allow recognition of the putative HZ/Mn interaction. However, as with the dose-response and long-term experiment, HZ did not have a significant effect on either maternal or fetal tissue Mn concentrations. Our observation that HZ feeding does not result in an induced Mn deficiency is consistent with the report that the

201

in vitro teratogenic effects of HZ were not alleviated by addition of Mn to the growth media 1131. (It should be noted that in the present study eight fetuses from each of the dietary groups in experiment 3 were examined for soft tissue abnormalities and an additional eight fetuses per group were examined for skeletal defects. All fetuses examined were judged to be normal independent of drug or dietary Mn group.) Consistent with the present work, Sakamoto [14] reported that long-term injection of HZ in both control and Mn-deficient mice had no effect on tissue Mn concentrations as measured by neutron activation analysis as we11as excretion of 54Mn. Furthermore, this investigator suggested that although Mn has been reported to alleviate some HZ-induced side effects [2,6], potential biochemical explanations for this effect have not been identified. Although HZ-induced changes in tissue Mn levels were not observed, kidney Cu concentrations were higher in all groups of animals receiving HZ in their diet compared to controls (Table I-III). Dose-response results showed a 2-fold increase in kidney Cu levels after only 2 weeks of HZ feeding. Results from the long-term and 21-day pregnancy experiment were similar in that there were 2- to 3-fold increases in kidney Cu concentrations in rats which received HZ in their diet compared to controls. These results are consistent with the finding that in humans, HZ therapy results in increased urinary excretion of Cu [lo]. We suggest that the HZ-induced increase in kidney Cu concentrations may reflect the stimulation of ceruloplasmin synthesis by the liver as an acute phase response to drug-induced tissue damage [ 151. An increase in acute phase protein synthesis may occur in response to stress, infection, tissue damage and various chemical insults [15,16]. One of the characteristics of an acute phase response is an increased plasma ceruloplasmin level which results in an increased plasma Cu concentration. Although plasma Cu levels in our experiment were not significantly different, there was a trend within dietary groups of slightly higher plasma Cu concentrations in the HZ-fed groups (Tables II and III). Our observation that tissue Fe levels were significantly lower in HZ-treated groups is of interest due to the report by Rapaka et al. [13] that supplementing Fe to an in vitro culture system alleviated HZ-induced alterations in cultured embryonic chicken bone explants. Both of these observations suggest that HZ interferes with Fe metabolism. HZ has been shown to form complexes with Fe in vitro [17]; thus it is possible that the lower tissue Fe levels noted in the HZ-fed animals are due to a reduced bioavailability of Fe in the diet secondary to the formation of a HZ-Fe complex. It is interesting to note that while liver Fe levels were decreased by HZ feeding, liver Mn levels tended to increase. It is well known that Fe and Mn interact in bioiogical tissues [IS] and it is possible that the effects of HZ on liver Mn were in part due to the effects on liver Fe or vice versa. In summary, while it has been reported that Mn has a protective effect against HZ-induced side effects, the evidence presented in this paper as well as that of Sakamoto [14] shows that HZ does not affect tissue Mn levels. Thus, a HZ-induced

202

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