THE ANATOMICAL RECORD 196~183-190 (1980) Ultrastructural Effects of Riboflavin Deficiency on Rat Hepatic Mitochondria BERNARD TANDLER AND CHARLES L. HOPPEL Department of Oral Biology, School of Dentistry, and Departments ofPharrnncology and Medicine, School ofllledicine, Case Western Reserve University, and Veterans Administration Hospital, Cleweland, Ohio 44106. ABSTRACT The hepatic mitochondria of rats fed a riboflavin-deficient diet were examined by electron microscopy. Discoidal mitochondria with elongated cristae increased in frequency from day 4 to day 12 of the test diet. At day 22, sheaves of closely packed cristae were present in many otherwise typical mitochondria. At day 53, numerous cupshaped mitochondria appeared; these often nested one inside the other. From this day onward, the mitochondria showed a tendency towards increasing size. By day 82, some had a diameter greater than 8 pm. These and other, smaller mitochondria often contained extremely prominent matrix granules. The production by riboflavin-deficiency of giant mitochondria in the rat liver appears to be unrelated to their capacity to carry out oxidative metabolism. Because riboflavin is intimately associated with a variety of hepatic mitochondrial enzymes, a deficiency of this vitamin can have widespread repercussions in liver cell function and structure. This has been demonstrated in mouse hepatocytes, where ariboflavinosis results in marked generalized depression of mitochondrial oxidative metabolism (Hoppel and Tandler, '75) and in mitochondrial structural aberrations, principally t h e formation of megamitochondria (Tandler et al., '68). In a recent biochemical study of hepatic mitochondria from riboflavin-deficient rats (Hoppel et al., '79), it was found that oxidative metabolism was severely affected, but was substrate-specific in contrast to the generalized defect in the mouse. Although there is a degree of parallelism between the biochemistry of riboflavin deficiency in the rat and that in the mouse, the one full-scale ultrastructural study of hepatocytes in ariboflavinotic rats reported only minor morphological alterations of the mitochondria that i n no way resembled the drastic changes that occur in the mouse (Sugioka et al., '69). It was for this reason that we felt that a reinvestigation of the morphological effects of riboflavin deficiency on rat hepatocyte mitochondria was in order. Accordingly, rat livers were monitored by electron microscopy for up to 83 days of deficiency. We found that mitochondrial structural perturbations occurred quite early in the course of the diet, and that greatly 000-3276X/80/1962-0183$01.70 0 1980 ALAN R. LISS. INC. enlarged mitochondria appeared only after prolonged riboflavin deficiency. MATERIALS AND METHODS Male Wistar strain weanling rats (21 days old) were obtained from Canvorth Farms (New City, N.Y.). Control animals were fed Purina Rat Chow. Test animals were fed a similar diet that lacked only riboflavin (Nutritional Biochemical Corp., Cleveland, Ohio); the composition of the test diet was detailed in a previous report (Hoppel and Tandler, '75). Food and water were available ad libitum to both groups. An earlier investigation of the biochemistry of hepatic mitochondria in riboflavin-deficient rats showed that the various parameters studied were unaffected by variations in caloric intake (Hoppel et al., '79); for this reason pair-fed controls were not used in the present study. Riboflavin-deficient rats (a total of 36 experimental animals) were killed by decapitation after the following number of days on the test diet: 4,7,10,11,12,14,22,28,36,43,46,49,53, 56, 60,63,74,75,81,82, and 83. Specimens of liver were fixed for 2 hours at room temperature in phosphate-buffered POosmium tetroxide (Millonig, '61a). They were then rinsed in distilled water, dehydrated in ethanol, passed through propylene oxide, and embedded i n Received May 22, 1979; accepted August 14, 1979. 183 184 BERNARD TANDLER AND CHARLES L. HOPPEL Maraglas-DER 732 (Erlandson, '64). Thin sections were stained with methanolic uranyl acetate (Stempak and Ward, '64) and lead tartrate (Millonig, '61b) and examined in a Siemens Elmiskop la electron microscope. some showed modest enlargement, attaining diameters of about 3 pm. In these enlarged organelles, cristae tended to be restricted to the periphery, but a stack of appressed cristae often lay in the matrix compartment (Fig. 3). Mitochondria of bizarre shape were occasionally OBSERVATIONS noted. Branching and Y- or cup-shaped mitoAs early as the fourth day of the deficient chondria were common. Such mitochondrial diet, a few hepatic mitochondria exhibited configurations did not preclude the presence of structural alteration. Such organelles were stacked cristae-these membrane arrays ocelongated, measuring about 3 pm in length, curred in the odd-shaped mitochondria with the with an attenuated central region and bulbous same frequency as they did in typical organterminal expansions. In some of these mito- elles. Occasional cells contained a substanchondria, closely packed cristae ran longitudi- tially increased mitochondrial population. In nally in the bulbous portion. Mitochondria with these cells, the mitochondria generally lacked this apparent dumbbell shape were far more stacked cristae and, with the exception of a few common at the twelfth day of the diet, at which enlarged forms, were typical in appearance. At day 53, the mitochondria were larger and time they had increased somewhat in length. Serial section analysis demonstrated that these more abundant than previously. They showed a mitochondria actually were biconcave discs, marked propensity for nesting one inside the sometimes in the form of a cup. The attenuated other (Fig. 4, 5 , and 6). At day 60, occasional mitochondria exmidregions of such mitochondria, as seen in thin section, accommodated a single, fene- hibited a considerable degree of enlargement strated, longitudinally-oriented crista. These, (Fig. 7 ) .Many of these larger organelles had an and all subsequent mitochondrial alterations, irregular shape. This trend toward mitochonoccurred independently of hepatocyte position drial giantism was more obvious at day 7 5 . The large mitochondria tended to be spherical and within the liver lobules. At day 22, many mitochondria that were had diameters of - 4 pm. Regardless of size, a otherwise normal in size and appearance con- number of mitochondria contained stacks of tained a stack of closely packed cristae (Fig. 1). cristae similar to those seen earlier (Fig. 8 and The number of cristae in such stacks ranged 9). By day 82, mitochondrial enlargement from two to more than a dozen." The orienta- reached its maximum (Fig. 10). The larger ortion of these stacks varied from mitochondrion ganelles averaged about 5 pm in diameter, but t o mitochondrion. In some organelles, the some attained a diameter greater than 8 pm. stacked membranes were transversely ori- The matrix granules were extremely promiented, while in others they were obliquely dis- nent in many organelles, measuring up to 160 posed. In still other mitochondria, especially in nm in diameter (Fig. 11).A few mitochondria the more elongated variety, the cristal stacks contained small stacks of appressed cristae; usually were associated with one end of the these seemed to be involved in the formation of organelle, where they were longitudinally ori- intramitochondrial ;.acuoles by progressive diented. Occasionally, some of these longitudinal latation, as shown in Figure 12. The net result cristae were quite long, extending almost the of this process was the presence in some enlarged mitochondria of serried vacuoles conentire length of the rod-shaped mitochondrion. While in most mitochondria in which they taining variable amounts of a flocculent, occurred the stacked cristae were separate and moderately dense material. distinct, in certain of these organelles the intervals between adjacent cristae were obliterated, so that neighboring membranes were closely appressed, giving them a n unusually *During the review process it was suggested by one of the reviewers dense appearance. A few cristal stacks con- that the manner in which the stacked membranes are associated with sisted entirely of these appressed cristae, but the inner mitochondrial membrane could be established by serial section analysis. W i l e the point is well taken, it was not feasible in the other stacks were composedin part of appressed present case, since the tissue specimens were embedded in Maraglascristae and in part of individually discernible D.E.R. 732, a material that only rarely permits ribboning of sections. The information that could be obtained by repeating the experiment cristae (Fig. 2). with a more suitable embedding medium would not significantly affect Although most mitochondria in the 22-day our conclusions, and for this reason we elected not to repeat the experiment since. in our orinion. the time & exuense would not be iustified deficient ;ats were in the normal size range, Fig. 1. Mitochondria with stacked cristae. 22-day riboflavin-deficient rat x 26,125. Fig. 2. A mitochondrion with two stacks of appressed cristae. The close packing of the cristal membranes produces an apparent increase in membrane density. 22-day riboflavin-deficient rat. x 47,500. Fig. 9. An enlarged mitochondrion containing a stack of cristae. 22-day riboflavin-deficient rat. x 24,700. 186 BERNARD TANDLER AND CHARLES L. HOPPEL Fig. 4. Two sets of nested mitochondria. In this plane of section, spherical mitochondria appear to be surrounded by ringshaped organelles. 53-day riboflavin-deficient rat. x 23,000. Fig. 5. An aggregation of mitochondria that seems to consist of concentric ringshaped organelles. Elements of RER are present between the adjacent mitochondria. 53-day riboflavin-deficient rat. x 14,500. Fig. 6. A pair of closely apposed mitochondria in which a rod-shaped organelle is seated in a cupshaped one. Should the plane of section pass through the rim of such an aggregate, it would yield the organelle relationship shown in Figure 4.53day riboflavin-deficient rat. x 21,000. HEPATIC MITOCHONDRIA IN RlBOFLAVIN DEFICIENCY 187 Fig. 7 . A substantially enlarged, irregularly shaped mitochondrion. 60-day riboflavin-deficient rat. x 21,850. Fig. 8. An enlarged mitochondrion containing a stack of cristae. Several of the cristae are unusually electron-dense. 75-day riboflavin-deficient rat. x 20,900. Fig. 9. A portion of the preceding electron micrograph a t higher magnification. It is evident that the increased density of certain of the cristae is due to their dense content. x 95,000. 188 BERNARD TANDLER AND CHARLES L. HOPPEL Fig, 10. Survey micrograph of a hepatocyte in an 82-day riboflavin-deficient rat. Several of the mitochondria are greatly enlarged; the one at the right almost matches the nucleus in size. X 9,025. Fig. 11. An enlarged mitochondrion containing extremely prominent matrix granules. 82-day riboflavin-deficient rat. x 16,150. Fig. 12. An enlarged mitochondrion with several contiguous dilated cristae that form a rank of electron-lucentvacuoles. 82day riboflavin-deficient rat. x 10,450. HEPATIC MITOCHONDRIA IN RIBOFLAVIN DEFICIENCY DISCUSSION Our observations indicate that riboflavin deficiency in rats has more significant ultrastructural consequences than was previously recognized. We have confirmed that ariboflavinosis results in the formation of stacks of cristae in liver mitochondria. However, these cristal aggregates appear at a considerably earlier time and persist longer t h a n was reported by Sugioka et al. ('69). In some mitochondria with stacked cristae, adjacent membranes appeared to be fusing; a similar process in ariboflavinatic mouse hepatic mitochondria leads to the formation of myelin figures, a sign of organelle degeneration. In the rat, however, we observed relatively few mitochondria in autophagic vacuoles. We found that rats fed a riboflavin-deficient diet for 6@83 days developed giant hepatic mitochondria. Luse et al. ('62) previously described enlarged mitochondria with rarified matrices in 8-week riboflavin-deficient rats. Because of inadequate preparative techniques used by these workers, e.g., methacrylate embedment, the increase in mitochondrial size a s well as the matrix pallor may have been due in part to swelling of these organelles. Similarly, although Taniguchi et al. ('78) reported a degree of mitochondrial enlargement in mitochondria of rats fed a riboflavin-deficient diet for five weeks, the disrupted limiting membranes seen in their illustrations bespeaks swelling rather than true size increase. In contrast to these findings, the enlarged mitochondria we observed had a substantial matrix density that precisely matched that of neighboring normal-sized mitochondria; mitochondrial membranes were always intact. The manner in which mitochondrial enlargement was accomplished in the rat was not determined. Tandler et al. ('68)have speculated that in the mouse, where riboflavin deficiency of six weeks duration leads to the formation of giant mitochondria, some bigger than the hepatocyte nuclei, this increase was brought about by fusion of smaller organelles and, to a lesser extent, by growth of the mitochondrial mass. Histometric analysis of vitamin B,-deficient mouse liver cells by Rohr et al. ('74) has confirmed that both these mechanisms in mitochondrial enlargement are in fact operative. Some cells i n our study had increased numbers of mitochondria. Luse et al. ('62) stated that hepatic mitochondria are more abundant i n 8-week riboflavin-deficient rats, but did not allude to the sporadic nature of this organelle enhancement. In contrast, Reith et al. ('73) re- 189 ported that quantitative histometric analysis of liver cells from 83-day riboflavin-deficient rats shows a decrease in the number of mitochondria and a concomitant reduction in the mitochondrial volume fraction. Using a n electronic particle counter, Scarpelli et al. ('71) found that the hepatic mitochondrial population was virtually halved after three weeks of ariboflavinosis in the rat; however, as these workers themselves admit, the built-in limitations associated with this methodology may have introduced serious errors into these measurements and the results of electronic particle counting of isolated mitochondria must be considered semiquantitative at best. We previously reported that addition of the riboflavin analogue, galactoflavin, to a riboflavin-deficient diet greatly speeds the appearance of the morphological symptoms of riboflavin deficiency in mouse liver cells (Tandler and Hoppel, '74). Giant mitochondria appear by the 13th day, rather than a t the 42nd day as is the case in simple vitamin B, deficiency. The cristae in the galactoflavin-supplemented hepatic megamitochondria tend to be peripheral, rather than being evenly distributed as they are in the giant mitochondria of simple deficiency. In this respect, the enlarged hepatic mitochondria in our riboflavin-deficient rats more closely resembled the megamitochondria of galactoflavin-supplemented mice than they did those in mice made ariboflavinotic by simple dietary deficiency. Norton et al. ('77), who investigated the effects of galactoflavin-induced riboflavin deficiency on the ultrastructure of rat liver cells, state that galactoflavin produced no abnormalities in mitochondrial structure. This absence of an effect on the mitochondria is probably due to the fact that Norton et al. used adult rats in their studies. In our experience, adult rodents are relatively refractory to manipulation of dietary riboflavin, and only weanling animals are wholly responsive to such treatment (Tandler and Hoppel, '72; Hoppel and Tandler, '78). In mice made ariboflavinoticby either simple riboflavin deficiency or by galactoflavin-supplementation, mitochondrial oxidative metabolism is severely decreased; these alterations occur in precisely the same time frame in both conditions (Hoppel and Tandler, '76). However, megamitochondria appear a t day 13in galactoflavin-supplemented mice and a t 42 days in simple riboflavin deficiency. In contrast to mice, simple riboflavin deficiency in rats does not result in a generalized impairment of oxi- 190 BERNARD TANDLER AND CHARLES L. HOPPEL dative metabolism; instead, the deleterious effects of this diet are substrate-specific. For example, fatty acid oxidation shows a marked decrease within 24 hours after initiation of the deficient diet, whereas a-ketoglutarate or p p v a t e metabolism is not affected even with prolonged deficiency. While the major changes that occur in mitochondrial oxidative metabolism are rapidly manifested, giant mitochondria make their appearance in rat liver quite late in the deficient state (day 60). Based on our observations both in the mouse and rat, the genesis of megamitochondria is unrelated to the oxidative status of the hepatic mitochondria. The production of these enlarged organelles depends on one or more undetermined factors, perhaps involving mitochondrial membrane phospholipids. We are exploring this possibility with additional studies. ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grants AM-15804 and 5 SO7 FtR 05335, and the Medical Research Service of the Veterans Administration. LITERATURE CITED Erlandson, R.A. (1964) A new Maraglas-D.E.R. 732, embedment for electron microscopy. J. Cell Biol., 22:704-709. Hoppel, C., J.P. DiMarco, and B. Tandler (1979) Riboflavin and rat hepatic structure and function. 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