inhibition of mitochondrial respiration by cationic rhodamines as a possible teratogenicity...

TOXICOLOGY AND APPLIED PHARMACOLOGY 99,8 1-89 (1989)

Inhibition of Mitochondrial Respiration by Cationic Rhodamines as a Possible Teratogenicity Mechanism’

SULABHA RANGANATHAN, PERRY F. CHURCHILL, AND RONALD I). HOOD*

Department ofBiology, The University ofAlabama. Tuscaloosa, iilabama 35487-0344

Received June 20. 1988; accepted January 4, I989

Inhibition of Mitochondrial Respiration by Cationic Rhodamines as a Possible Teratogenicity Mechanism. RANGANATHAN, S., CHURCHILL, P. F., AND HOOD, R. D. (1989). Toxicol. Appl. PharmacoL.99,8 l-89. Exposure of mice to cationic rhodamines, Rh 123 and Rh 6G, has been found to be associated with developmental toxicity, while neutral rhodamines (e.g.. Rh B) had no such effect. When mouse embryos from dams given ip injections of Rh 123, Rh 6G ( I5 mg/ kg), or Rh B (30 mg/kg) on gestation Day (GD) 10 were examined, Rh 123 and Rh 6G were present in embryonic tissue in fluorescent bodies within the average dimensions of mitochondria. Rh B was evenly distributed in the cytoplasm. With in vitro exposure of isolated mitochondria to rhodamines on CD 12,3-4 times more Rh 123 was associated with mitochondria under energized conditions than under nonenergized conditions; the amount of Rh 6G associated with mitochondtia was much less under either condition. Treatment of pregnant mice (ip) with Rh 123 (15 mg/kg/day) or Rh 6G (0.5 mg/kg/day) on GD 7-10 resulted in inhibition of state 3 respiration of embryonic mitochondria isolated on GD 12. When isolated embryonic mitochondria were exposed to the cationic rhodamines, inhibition of state 3 respiration was dose dependent. With 5 pg of Rh 123/mg mitochondrial protein, state 3 respiration decreased by 31%. while Rh 6G (1 rg/mg) decreased state 3 respiration by 27%. In vivo exposure of maternal liver mitochondria to cationic rhodamines did not result in inhibition of respiration 2 days later, whereas in vitro results were similar to those for embryonic mitochondria. In vivo or in vitro exposure to Rh B had no effects on mitochondrial respiration. These results indicate that interference with embryonic energy metabolism is a possible mechanism by which cationic rhodamines exert adverse effects on embryogenesis. $1 1989 Academic Press, Inc.

Cationic fluorescent rhodamines selectively accumulate in mitochondria of living cells, whereas neutral rhodamines do not (Johnson et al., 1981). Even though cationic rhodamines are positively charged at physiological pH, the charge is distributed over their aro- matic rings and thus does not interfere with their lipophilic nature. Once these cationic rhodamines enter mitochondria, they tend to become trapped due to the high negative elec- trical potential across the mitochondrial membrane (Johnson et al., 1980, 1981; Em-

aus et al., 1986), and dissipation of the transmembrane potential of isolated mitochondria by ionophores or electron transport in- hibitors eliminates selective mitochondrial uptake of these compounds (Johnson et al.. 198 1). Other studies with human fibroblasts and human breast adenocarcinoma cells have confirmed these results (Goldstein and Korczack, 198 1; Davis et al., 1985).

Rhodamine 123 (Rh 123), although cationic, is of low toxicity to many types of cells. Nevertheless, in vitro studies done with newborn rat cardiac muscle cells, which have

’ This work was supported in part by PHS Grant high plasma transmembrane potentials, have BRSG 1751-08. shown that Rh 123 is retained for prolonged

’ To whom reprint requests should be addressed. periods in the mitochondria, inhibits con-

81 0041-008X/89 $3.00 Copyright Q 1989 by Academic Press. Inc. All rights ofrep~‘oductmn in any form reserved.

82 RANGANATHAN, CHURCHILL. AND HOOD

tractions, and results in cell death (Summer- hayes et al., 1982; Lampidis et al., 1984). Rh 123 and another cationic dye, rhodamine 6G (Rh 6G), both inhibit oxidative phosphorylation in rat liver mitochondria (Gear, 1974; Higuti et al., 1980; Mai and Allison. 1983; Modica-Napolitano et al., 1984; Modica-Na- politano and Aprille, 1987). Rh 123 is also retained for long periods by carcinoma cells and exhibits antitumor activity both in vitro and in vivo (Bernal et al.. 1982, 1983; Lam- pidis et al., 1983; Abou-Khalil et al., 1985; Beckman et al.. 1987; Castro et al.. 1987; Modica-Napolitano and Aprille, 1987).

In order to investigate the potential of cationic rhodamines to induce mammalian developmental toxicity and to determine their effects on embryonic energy metabolism, we chose the mouse as a model. Previous studies performed to exploit the availability of struc- turally related compounds that differ greatly in their biological effects have shown that both Rh 123 and Rh 6G could induce developmental toxicity. including malformations, when pregnant mice were treated with these agents during mid-gestation (Jones et al.. 1986: Hood et al., 1988). Rhodamine B (Rh B) and rhodamine 116 (Rh 116), both neutral dyes, failed to show such effects on embryos at doses similar to those used for the cationic dyes. Rh 123 can also interfere with the development of Drosophila melanoguster (Zhang and Hood, 1987).

In the current study, mouse embryos were examined for the presence and localization of rhodamine dyes in order to determine whether rhodamines may enter and thus di- rectly affect embryonic tissues. Cationic rhodamines were found to enter embryonic cells and to be selectively taken up by mitochondria, while a neutral rhodamine failed to ex- hibit such specific uptake. Both in vivo and in vitro effects of rhodamines on mitochondrial respiration were studied. Rh 123 and Rh 6G were found to inhibit mitochondrial respiration following both in vivo and in vitro expo- sures, while a neutral rhodamine failed to inhibit respiration at similar exposure levels.

METHODS

Female CD-l strain mice (26-35 g) were obtained

from Charles River Breeding Labs (Raleigh, NC) and housed for mating with experienced males (35-45 g) of

the same strain. A 12/12 hr light/dark cycle was used. Temperature was maintained at 22 f 2°C. with 40-60s relative humidity. Food (Purina Lab Chow, Purina Mills.

Richmond. IN) and water were provided ad lihitum.

Rhodamines (laser grade) were purchased from Eastman Kodak Organic Chemicals (Rochester, NY), and solutions were prepared in deionized distilled water.

ADP was purchased from Sigma Chemical Co. (St. Louis, MO).

@V /ocukafion. Pregnant mice were treated ip with

Rh 123 (15 mg/kg), Rh 6G (15 mg/kg), or Rh B (30 mg/

kg) on gestation Day 10 (copulation plug = Day I). Two hours later, the mice were killed and their embryos were removed. Frozen I- to 2-pm-thick sections through the

embryos were made with a cryotome and examined for the presence of rhodamines by epifluorescence micros-

copy. Maternal liver tissue was similarly examined for the presence of the dyes.

Isolation (?i mitochondria. For in ,aivo experiments,

pregnant mice were treated with Rh B or Rh 123 ( 15 mg/ kg/day) or with Rh 6G (0.5 mg/kg/day) on gestation

Days 7- 10. Control mice and mice to be used in in vitro

experiments were left untreated. The mice were killed on

gestation Day 12. and their embryos as well as the maternal livers were transferred to buffer containing 0.25 M

sucrose. IO mM Tris-HCI. and 1 mM EDTA. pH 7.5. at

0°C. Mitochondria were isolated from pooled embryos and maternal liver by the method of Bargman rr ul.

( 1972). All preparations were maintained at 0°C. Tissues were homogenized with a motor-driven homogenizer

with a Teflon pestle and centrifuged at SOOg for 15 min. The supernatants were centrifuged again at 11,OOOg for 15 min. Because quantities of embryonic material were

limited, the resultant pellets from embryos were used without further purification. Maternal liver mitochondria were further purified by another centrifugation

( 1 1,OOOg for 15 min). Mitochondrial protein concentrations were determined by use of bicinchoninic acid (Smith PI n/.. 1985). with bovine serum albumin as the

standard. @untifiution o/‘Rh 123 und Rh hG as.cocr~~trd with

mifochondriu This was done by a modification of the method of Higuti (‘1 ol ( 1979). Mitochondria from untreated embryos were incubated at room temperature for 5 min in I ml of respiratory medium (see below), with or

without 10 pg DNP. in the presence of 10 pmol succinate. 100 nmol ADP. and rhodamines in microgram quantities. The reaction mixtures were cooled rapidly to 0°C by transferring the tubes to an ice bath and were centrifuged at 8000,~ for 2 min in a microcentrifuge. The

amount of Rh 123 in the supernatant was determined by absorbance at 502 nm in comparison with standards.

INHIBITION OF MITOCHONDRIA BY RHODAMINES 83

The amounts of Rh 123 associated with mitochondria

were determined by subtracting the quantity measured in the supernatant from the amount of the dye originally

added. Rh 6G was determined in the same way, at a

wavelength of 527 nm. Mitochondriul respiration. Oxygen uptake by mito-

chondria was measured polarographically at 37°C. as described by Estabrook (1967) with sodium succinate as substrate. Instrumentation consisted of a Yellow Springs

Instrument Co. (Yellow Springs, OH) Model 5300 biological oxygen monitor and Model 5357 micro oxygen

probe and an Instech Laboratories (Horsham, PA) Model 600B micro oxygen chamber. Respiratory me-

dium contained 225 mM sucrose. 1 mM EDTA. 10 mM K2HP04-KH?PO?. 5 mM MgCh. and 10 mM Tri-HCl.

pH 7.4 (Aprille and Asimakis, 1980). Total oxygen con- tent of aerated buffer in the O.&ml chamber was determined by the method of Chappel ( 1964).

In order to measure the influence of in vivo rhodamine treatment on embryos. mitochondria from embryos tahen from dams that had been injected as described

above with Rh 123. Rh 6G, or Rh B during gestation were introduced into the chamber containing respiratory

medium. After 1-2 min, succinate was added to the chamber to initiate state 4 respiration. After state 4 respi-

ration was assessed for l-1 min. 100 nmol ADP was added to the medium. and state 3 respiration was measured. Uncoupler-stimulated respiration was measured

after addition of t-2 peg DNP. Mitochondria from untreated embryos were used as controls.

To measure the effect of in rirro treatment with rhodamines. mitochondria isolated from untreated mouse em-

bnos were introduced as above into the chamber containing respiratory medium. Rhodamines were added to the medium in microgram quantities. State 3 and state

4 respirations. as well as uncoupler-induced respiration. were assessed as described above. Respiratory control ra-

tios (RCR). defined as the ratio of oxygen consumption in the presence and absence of ADP when substrate concentration is not limiting(Cain and Skilleter. 1987). were

determined for both in vitro and in viva trials.

.S/nti.s~ic~/ u~n~/j~.si.s. The differences associated with treatments were analyzed by a two-tailed Student’s t test

(Iman and Conover. 1983).

RESULTS

IIJV loculixtion. When embryonic sections were examined 2 hr after dye exposure for the presence of rhodamines on gestation Day 10, Rh 123 was present in highly fluorescent bodies (Fig. l), and these were within the range (0.2-3 pm diameter and 7-10 pm length) of average dimensions of mitochon-

dria (Threadgold, 1976). An identical distri- bution was found for Rh 6G, which was present in apparently lesser amounts. In the case of Rh B, embryonic tissue failed to show lo- calized fluorescence. Maternal Rh B dosage with twice the amount used in the case of the cationic dyes was required to obtain sufficient fluorescence to detect in the embryonic tissue, and the neutral dye was evenly distributed in the cytoplasm (Fig. 2). Similar distri- butions of cationic and neutral rhodamines were observed in maternal liver sections.

.4ssociutioe with mitochondria. Localiza- tion of cationic rhodamines in mitochondria was also evidenced by spectrophotometric measurement (Fig. 3). Approximately 3-4 times more Rh 123 was associated with energized mitochondria than was the case under nonenergized conditions. The amount of Rh 6G associated with mitochondria was much less than that of Rh 123 under similar conditions, and Rh 6G association with energized mitochondria was only 1.5 to 2 times greater than that observed for nonenergized mitochondria.

Effects on mitochondriar’ respiration. The influence of cationic rhodamines on mitochondrial respiration can be seen in a typical oxygen electrode trace (Fig. 4). Addition of 100 nmol of ADP to coupled mitochondria respiring with succinate as substrate increased the rate of oxygen consumption from 52 to 19 I ng atoms/mg protein/min. When 5 wg of Rh 123 was added to the medium, State 4 respiration was increased from a level of 52 to one of 7 1 ng atoms/mg protein/min. State 3 (ADP dependent) respiration was inhibited. decreasing from a level of 191 to one of 128 ng atoms/mg protein/min. A dose-related inhibition of state 3 respiration was observed over the range of Rh 133 concentrations used (Table 1). Also. RCR values decreased. further indicating an inhibita’ry effect on mitochondrial function. At conlcentrations above 8 pg Rh 123/mg protein, state 3 and state 4 respiration could not be distinguished. State 4 respiration increased in a dose-dependent


FIG. Fll I5 mg/kg maternal dose of Rh 123 (X 1400).

uorescence micrograph of section of gestation Day 10 mouse embryo after in vi\ rxposure to

manner, indicating possible uncoupling by the dye.

Rh 6G caused a greater inhibition of state 3 respiration at a lower in vitro concentration than did Rh 123. However, Rh 6G had no significant effect on state 4 respiration at concentrations in the medium sufficient to inhibit state 3 respiration. Uncoupler-stimulated respiration increased following addition of either Rh 123 or Rh 6G; however, this in- crease was greater in the presence of Rh 123. At the 8 pg level of Rh 123, uncoupler-stimulated respiration was inhibited, and this concentration of dye may have resulted in a gen- eral inhibition of electron transport. Rh B, on the other hand, did not significantly affect state 3 or state 4 respiration or uncoupled res-

piration when compared to the untreated controls.

When mice were treated in vivo with known teratogenic doses of either cationic rhodamine. significant inhibition of state 3 respiration was seen in their isolated mitochondria (Fig. 5). The cationic rhodamines, and especially Rh 123, appeared to have stimulated state 4 respiration, but the effect was not statistically significant. Thus, the increased state 4 respiration and presumptive mitochondrial uncoupling seen at the higher Rh 123 in vitro exposure levels may not be a relevant factor in Rh 123-induced developmental toxicity. Levels of both state 3 and state 4 respiration for Rh B-treated embryos were close to control values.


FIG. 2. Fluorescence micrograph of section of gestation Day 10 mouse embryo after in viva exposure to 30 mg/kg maternal dose of Rh B (X 1400).

When mitochondria from maternal livers were tested following rhodamine treatment, in rive preparations failed to show significant inhibition of mitochondrial function, whereas in vitro preparations exhibited results that were very similar to those from the in vitro exposed embryonic mitochondria.

DISCUSSION

In our previous studies. Rh 123 (8 to 15 mg/kg/day) plus 2-deoxyglucose (500 mg/kg/ day), an inhibitor of glycolysis, were adminis- tered to pregnant mice on gestation Days 7- 10. Treatment was associated with malformations. such as exencephaly, omphalocele,

and meromelia (Jones et al., 1986; Hood et at., 1988). Rh 6G given on gestation Days 7- 10 caused maternal lethality at doses as low as 2 mg/kg/day and was embryo lethal at 1 mg/kg/day. Even at 0.5 mg/kg/day, Rh 6G induced developmental toxicity, including malformations. Rh B and Rh 1 16 generally failed to cause adverse effects, even at 15 mg/ kg/day for the same 4 days. Also, 2-deoxyglu- case alone was not associated with adverse effects except for retarded skeletal ossification (Jones et al., 1986; Hood et al.. 1988).

Localization of cationic rhodamines in embryonic cells in bodies that appeared to be mitochondria indicates that at least part of the adverse effect on the conceptus may be a direct influence on embryonic metabolism.

86 RANGANATHAN. CHURCHILL. AND HOOD

160 r

Amount of Rhodamme Added (jqlmg protein)

FIG. 3. The amounts of cationic rhodamines associ-

ated with mitochondria under energized and nonenergized conditions. Each value is a mean h SD of three to four experiments. Values for Rh 123 uptakes under

energized (0) vs nonenergized (A) conditions were significantly different from each other at all concentrations

tested (p < 0.0 I ). Similar comparisons for Rh 6G under energized (0) and nonenergized (a) conditions were not

significant (p > 0.05). Differences between the two compounds were significant at all concentrations under energized conditions and only at I5 and 20 pg/mg levels un-

der nonenergized conditions (I, < 0.05).

This was further supported by the in vitro data where cationic rhodamines were shown to be associated with embryonic mitochondria. The results also indicated that rhodamine uptake in the embryo was an energy- dependent process. Similar energy-dependent rhodamine uptake has been reported in isolated rat liver mitochondria (Higuti el al.. 1980; Modica-Napolitano et al.. 1984).

When mouse embryo or maternal liver mitochondria were treated in vitro with cationic rhodamines, the dyes caused significant inhibition of mitochondrial respiration. Similar findings were reported by others for isolated mitochondria of several types, including those from cells of rat liver (Gear, 1974; Hi- guti et al., 1980; Modica-Napolitano ef ul.. 1984) Friend leukemia (Abou-Khalil ez al.. 1985), human colon carcinoma (Modica-Na- politano and Aprille, 1987). and newborn rat myocardium (Lampidis et al.. 1984).

Mitochondria from embryos exposed in vivo also showed significant inhibition by cationic rhodamines. The level of inhibition of state 3 respiration found after in viva treatment with Rh 123 ( 15 mg/kg/day) was com- parable to that seen following in vitru exposure to Rh 123 levels of 2 and 5 pg/mg mitochondrial protein. Similarly, for Rh 6G, in vivo treatment (0.5 mg/kg/day) resulted in a degree of inhibition similar to that seen following in vitro exposure of mitochondria to 1 pg/mg protein. An important aspect of these data is this correlation between the in viva and in vitro data. Such a similarity between in viva and in vitro results further supports the possibility that rhodamines might cause developmental toxicity by inhibiting embryonic energy metabolism.

The fact that inhibition could still be seen in mitochondria on gestation Day 12. 2 days after the end of in viva dye treatment, indicates that the effect was long lasting, as was suggested by Lampidis et al. ( 1984) for newborn rat cardiac muscle. Also, the fact that in viva dye-exposed maternal liver mitochondria failed to show significantly inhibited respiration, in contrast to in vitro treated preparations. further supports the possibility that Rh 123 and Rh 6G may cause developmental toxicity by a direct influence on the embryo, rather than because of maternal toxicity.

FIG. 4. Typical oxygen electrode trace showing the effect of in vitro treatment with Rh 123 on mitochondrial respiration of Day 12 mouse embryos.


TABLE 1

INFLUENCEOFIN VITRO TREATMENTWITHRHODAMINEDYESONMOUSEEMBRYONIC

MIT~CHONDRIALRESPIRATION'

Dye

frglmg Protein)

Control 0

Rh 123 2

Rh123 5 Rh 123 8

Rh6G I Rh6G 2

RhB 5

State 3 respiration

(ngatoms/mg protein)

188.0 f 7.6

161.7 f 8.2

130.0+ 6.1 11 1.3 k 6.2

136.8 f 4.9 117.6 zk 5.2

183.1 k 9.1

State 4 respiration


53.0 f 5.7

59.2 f 5.9

70.0 -+ 4.8

75.7 f 7.2

55.5 + 5.3

57.6 +- 5.6

54.6 f 4.1

RCRh

3.55

2.73 1.86

1.47

2.46

2.04

3.35

Uncoupler stimulated respiration


128.5 + 9.6

150.1 k 8.3 167.2 k 9.7

97.6 + 5.6

134.8 f 4.2

143.2 + 8.9

131.7 f 8.8

’ Each value is a mean + SD of three experiments.

h Respiratory control ratio.

Relatively little information is available re- garding formation. development, and function of mitochondria during mouse embryogenesis. Leese and Barton ( 1984) have re-

COfltrOl Rh B Rh 123 Rh 6G

FIG. 5. Effect of in vivo treatment with cationic rhodamines (Rh 123, Rh 6G) and a neutral rhodamine (Rh B)

on mitochondrial respiration of Day 12 mouse embryos. Each value is a mean + SD of three to four experiments. State 3 respiration values of control and cationic rhodamines differed significantly (p < 0.005) but those of controls and neutral rhodamines did not (p > 0.05). Differ-

ences in state 4 respiration between control and treated groups were not significant (p > 0.05).

ported that glucose is the major substrate in unfertilized and fertilized ova and in the developmental stages up to the blastocyst. Benos and Balaban (1983) have shown that over 85% of ATP production in mouse blastocysts is via mitochondrial function: with glucose as the sole exogenous substrate, aero- bic respiration in blastocysts is increased by a factor of 14. The use of in vitro cultures of mouse embryos has indicated that glucose is the only energy source for early postimpian- tation embryos (Clough and Whittingham, 1983). Clough ( 1985) reported that during early somitogenesis, TCA cycle activity in- creases in rat and mouse embryos, and by the 25-somite stage, about half of the ATP equiv- alents of glucose catabolism could be from this pathway.

In the following discussion, the day on which a copulation plug was observed is con- sidered as gestation Day 1. It has been reported that embryonic rat heart sections show well developed outer mitochondrial membranes but poorly developed cristae by Day 11 of gestation. Gradual development takes place until Day 15, when mitochondria appear mature and nearly resemble adult heart mitochondria (Mackler et al., 197 1). It was also reported that mitochondria isolated


from rat embryos between Days 12 and 15 of gestation express an equal capacity for oxidative phosphorylation (Mackler et al., 1973). Robkin and Cockroft (1978) reported increased glucose and lactate production plus a decreased growth rate when rat embryos were exposed in vitro to lowered oxygen or ele- vated carbon monoxide levels on Days 12- 13 of gestation. Chloramphenicol and thiam- phenicol inhibited mitochondrial protein synthesis and caused increased resorptions and growth retardation following treatment of rats during organogenesis (Bass, 1975; Mackler et al., 1975: Oerter and Bass, 1975).

In mice, mitochondria appear to accumulate cationic rhodamines by gestation Day 10. Therefore, rhodamines may be interfering with embryonic mitochondrial function at that time. Current results also show significant inhibition of mitochondrial respiration which is an indirect indicator of oxidative phosphorylation in well-coupled mitochondria, on gestation Day 12 (the available amounts of mitochondrial protein on earlier gestation days were insufficient for direct as- say of mitochondrial function). Thus, these data support the possibility that interference with mitochondrial energy production may be at least in part the mechanism responsible for the developmental toxicity of cationic rhodamines in the mouse.

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