Testicular toxicity of the transferrin binding radionuclide 114min in adult and neonatal rats

Reproductive Toxicology, Vol. 9, No. 3, pp. 297-305, 1995 Copyright 0 1995Elsevier Science Ltd Printed in the USA. Allrights reserved

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TESTICULAR TOXICITY OF THE TRANSFERRIN BINDING RADIONUCLIDE 114mInIN ADULT AND NEONATAL RATS KATHARINE P. HOYES,* CLARE JOFINSON,? RUTH E. JOHNSTON,? ROGERG. LENDON,~JOLYONH.HENDRY,* HARRANSL. SHARMA,$

and IAN D. MORRISt *Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research,

Manchester, UK; tSchoo1 of Biological Sciences and *Department Biophysics, University of Manchester, Manchester, UK

of Medical

Abstract - Adult (70 d) and neonatal (7 d) male rats were dosed (i.p.) with 37 MBqlkg (1 mCi/kg; approximately 1 pg elemental indium/kg) 114mIn, a transfer&-binding radionuclide. In adults, approximately 0.25% of the injected activity localised within the testis by 48 b postinjection and remained constant for up to 63 d. In neonates, 0.06% of the activity was in the testis by 48 b, and this declined such that by 63 d only 0.03% remained. At 63 d, treated rats had reduced sperm head counts and abnormal testicular histology that was more marked in animals dosed as adults than as neonates. In vitro, uptake of 114mIninto seminiferous tubules isolated from 7-, 210-,or 70-d-old rats was compared with that of l”I. Both radionuclides were readily accumulated by the tubules. Whilst 114mInuptake into 20- and 70-d tubules was inhibited by excess traosferrin, uptake into 7-d tubules was unchanged. ‘=I uptake was not affected by excess transferrin. These data support the contention that some radionuclides may cross the blood-testis barrier by utilisation of the physiologic irontransferrin pathway, which may lead to greater testicular damage in adult compared to neonatal animals. Key Words: testis; spermatogenesis;

transferrin;

transferrin receptor; radionuclide; indium.

INTRODUCTION

taminants by the blood-testis barrier, formed by tight junctions between Sertoli cells at their basolateral surfaces (7). Our previous studies, however, have shown that the 114mInradionuclide can breach the blood-testis barrier and become associated with adluminal germ cells and their derived spermatozoa (8). Such testicular radionuclide localisation results in both spermatogenic and mutagenic damage (9). In common with many transition elements and some actinides such as plutonium (lo), americium, and curium (1 l), the indium ion binds strongly to the iron transport protein transferrin (12). All cells require iron as a constituent of a variety of biologic redox systems (13), and mammalian cells acquire this element through the receptor-mediated endocytosis of serum transferrin (14). Sperm-associated iron is constantly lost from the testicular iron pool; consequently there must be continuous transfer of iron from the general circulation to adluminal germ cells. This is achieved by a two-stage process in which serum transferrin is first internalised by receptor-mediated endocytosis at the basal pole of the Sertoli cell. The iron then dissociates from the transferrin and is bound by a second immunologically distinct testicular transferrin, synthesized within the Sertoli cell, whilst the serum transferrin

Epidemiologic data showing an increased incidence of childhood leukaemia in the offspring of employees of the nuclear industry (1,2) have reinforced existing concerns regarding the germ-line hazards presented by occupational, medical, and environmental radiation exposures. Whilst a variety of explanations have been invoked to explain these controversial findings (3,4), no firm concensus has yet been achieved regarding the mechanisms involved. Recent studies, however, have suggested that paternal preconceptional exposure to unsealed radionuclides may be a risk factor for childhood cancer (5). One possible explanation for these findings is that internal radionuclide contamination may lead to the localisation of radionuclides in the testis. Such local testicular exposure could result in the transmission of genetic damage via sperm irradiated during spermatogenesis (6). Germ cells in the seminiferous epithelium are protected from most toxicants and radioactive con-

Addresscorrespondenceto Dr. Katharine Hoyes, School of Biological Sciences, G.38. Stopford Building, University of Manchester, Oxford Road, Manchester, Ml3 9PT, UK. Received 9 August 1!)94; Revision received 7 November 1994; Accepted 7 November 1994. 297

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is recirculated back to the basal surface and released to the circulation (15). The diferric testicular transferrin is then secreted into the adluminal compartment where it binds to transferrin receptors expressed on the surface of adluminal germ cells and is internalised by receptor-mediated endocytosis (16). It is probable that this physiologic iron delivery pathway is the mechanism by which ‘14mIn and other transferrin-binding radionuclides can breach the blood-testis barrier and gain access to mutagenitally sensitive adluminal germ cells. Since the blood-testis barrier is established in the rat between 16 and 19 d of age (17), transferrin-binding radionuelides may behave differently after systemic administration to adult and neonatal animals. Therefore we have examined the in vivo distribution and testicular toxicity of 114mInafter intraperitoneal injection to 7-d-old and 70-d-old rats. In addition to these in vivo studies, we have also utilised an in vitro system of intact isolated rat seminiferous tubules (18). In this system, cell associations within the tubules are undisturbed, and tight junctions between Sertoli cells remain intact (18), thereby enabling closer study of the role of transferrin in testicular radionuclide localisation. The use of the 1’4mInradionuclide in these studies was prompted by both medical and environmental considerations. Indium radionuclides have widespread use in nuclear medicine for labelling blood cells, drugs, liposomes, and monoclonal antibodies. 1’4mIn-labelled lymphocytes have Furthermore, been used therapeutically to target malignant cells (19), and ‘14mInis of importance as an impurity in “IIn diagnostic radiopharmaceuticals. 1’4mIn also acts as a practical model isotope for the environmentally relevant alpha-emitting actinides such as plutonium, americium, and curium. The “4mIn radionuclide decays predominantly by orbital electron capture and internal conversion. These modes of decay are characterised by the emission of numerous low energy electrons with subcellular ranges from 0.01 to 7 pm. Consequently, the density of absorbed energy in the immediate vicinity of the decay site is very high and similar to that found along alpha particle tracks (20), and studies have shown that the radiotoxicity of DNA-bound Auger emitters is comparable to that of alpha-emitting radionuclides (2 1). MATERIALS

AND METHODS

Radionuclides 114mInwas prepared by neutron irradiation of enriched ‘131nat the Joint European Reactor (Pet-

Volume 9, Number 3, 1995

ten, The Netherlands). A typical irradiation for 1.5 mg ‘131nwas one reactor cycle of 25 d with a flux of 4.5 X lOI neutrons set-l cm-*. Specific activity at the end of neutron bombardment was 3.7 GBq/mg. The resultant l14”In was dissolved in 0.05N HCl to form indium chloride. lz51 (3.7 GBq/mL in NaOH solution) was purchased from Amersham International (Aylesbury, UK). Animals Adult male Sprague-Dawley rats (65 to 80 d old, 200 to 250 g) were purchased from Charles River Laboratories (Margate, UK). Neonatal (7 d old, 20 g) and immature (20 d old, 40 g) SpragueDawley rats were obtained from the University of Manchester Biological Services Unit. Neonates were kept with their dams until they were weaned at 18 d. Weaned and adult animals were housed 6 to a cage under normal laboratory conditions with free access to tap water and standard commercial diet. All animal studies were performed according to the requirements of the UK Animal (Scientific Procedures) Act of 1986. Biodistribution studies A total of 12 adult or neonatal rats were injected intraperitoneally with 37 MBq/kg (approximately 400 @Z/adult and 6 p Ci/neonate; corresponding to approximately 250 ng and 6 ng elemental indium respectively) ‘14mIndiluted in physiologic saline. At 48 h or 63 d postinjection, groups of 6 animals were weighed then killed by terminal anaesthesia and exsanguination. Testes, epididymides, vas deferens, prostate, kidney, liver, spleen, brain, and a piece of gastrocnemius muscle were dissected out, rinsed in saline, then blotted dry and transferred to preweighed plastic tubes. Approximately 0.5 mL of whole blood was removed by cardiac puncture. All tissues were weighed wet, then assayed for radioactivity using an Autogamma counter. All samples were counted under the same geometry, and activity measurements were corrected for background and physical decay. Organ activity is expressed both as the specific activity (i.e., the % injected activity/g tissue) and as the percentage of the injected activity per whole organ for those organs dissected whole. Testis histology and sperm head counts The left testis from each animal was fixed in Bouin’s solution overnight then stored in 70% ethanol. Testes were cut transversely to the long axis and subjected to routine histologic processing. Paraffin sections were cut at 3 pm and stained with

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ti4mIn in adult and neonatal testes 0 K. P. HOYES ET AL.

haematoxylin and eosin. A total of 200 tubules per animal were examined for spermatogenic lesions and recovery. All section were counted blind by a single observer. Right testes were stored at -20 “C until sperm heads were counted according to previously described methods (22).

vitamins (all from Gibco) with 5% foetal calf serum and buffered to pH 7.4 with 15 mM 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid (Hepes, BDH). To determine the effect of excess transferrin on radionuclide uptake, tubules were incubated with 37 kBq (1 ,uCi) n4mIn or lz51for 2 h in the presence of increasing concentrations of diferric human transferrin prepared as described previously (11). At the end of the experiment, tubules were washed three times with ice cold Hanks Balanced Salts Solution (HBSS) and treated with 0.5 mg/mL proteinase K for 30 min on ice to remove any surface-bound transfer-tin. The tubules were then washed twice more with HBSS and lysed with 1 N NaOH prior to gamma counting and protein determination.

Isolated tubule experiments

Seminiferous tubules were isolated from adult male rat testes by collagenase dispersion as described previously by Wauben-Penris et al. (18) and resuspended at approximately 1.5 mg protein/ml in incubation medium. The method used resulted in intact tubules with a plug of spermatozoa at each end that prevented substances such as trypan blue from entering the tubule. Tubules from 7-d-old and 20-d-old rats were found to be permeable to trypan blue after even very mild collagenase treatment. Therefore, testes from these animals were decapsulated then very gently teased apart with fine forceps prior to resuspension in incubation medium. This method resulted in intact tubules that were impermeable to trypan blue. Again the tubules were resuspended to approximately 1.5 mg protein/ml; this equated to one 7- or 20-d-old testis/ml. The isolated tubules were incubated at 34 “C in 1 mL Eagles Minimal Essential Medium (MEM) supplemented with L-glutamine (2 mM), sodium bicarbonate (0.85 g/L), nonessential amino acids, and Table 1. Tissue distribution

RESULTS Biodistribution

experiments

The biodistribution of 114mInin adult and neonatal rats at 2 and 63 d postinjection is shown in Table 1. To aid interpretation, the data are expressed both as organ-specific activity and as whole organ radionuclide uptake. At each timepoint, the highest accumulation of the radionuclide was observed in the liver, spleen, and kidneys in both adult and neonatal animals. At 2 d postinjection, however, 114mIn activity was noted in all resected tissues from both adult and

of i14mIn in adult and neonatal rats at 2 and 63 d postinjection 37 MBq/kg 114mIn

of

Percent injected activity/g tissue Adult Organ Testis Epididymis Vas deferens

Kidney Liver Spleen Brain Muscle Blood

Neonate

2d 0.18 (0.22 0.88 (0.33 1.63 (0.25 0.71 (0.12 0.74 (0.92 1.05 (16.78 1.24 (0.82 0.02 (0.02 0.09 0.14

f 0.028 * 0.02)b 2 0.23 k 0.09) f 0.18 * 0.03) 2 0.05 f 0.01) + 0.07 f 0.08) f 0.04 k 0.65) -r- 0.05 f 0.03) f 0.004 + 0.003) 2 0.02 k 0.01

63 d 0.33 (0.24 0.14 (0.12 0.05 (0.01 0.08 (0.05 0.37 (0.55 0.18 (3.06 0.9 (0.69 0.02 (0.02 0.09 0.01

f 2 2 t f * + f ” + + * * f 2 2 + 2

0.02 0.01) 0.02 0.01) 0.004 0.001) 0.01 0.01) 0.09 0.10) 0.03 0.45) 0.10 0.06) 0.003 0.005) 0.02 0.001

2d 4.57 2 0.65 (0.06 f 0.01) 5.39 f 0.87 (0.18 -+ 0.01) ND ND 10.54 (1.52 18.55 (16.72 8.61 (1.14 0.77 (0.37 1.45 1.69

5 1.04 k 0.15) f 2.01 2 1.23) ? 0.49 + 0.06) -r- 0.06 * 0.02) + 0.19 f 0.12

ND = Not determined. BMean + SEIM, n = 6. bValues in parentheses refer to the percentage of injected activity per whole organ.

63 d 0.02 (0.03 0.06 (0.03 0.06 (0.01 0.04 (0.01 0.11 (0.16 0.17 (3.01 0.26 (0.30 0.12 (0.18 0.02 0.003

k 0.01 -+ 0.01) + 0.02 2 0.01) + 0.004 + 0.004) + 0.01 f 0.005) + 0.02 f 0.02) + 0.03 + 0.5) + 0.04 + 0.03) + 0.04 2 0.04) f 0.005 -c 0.001

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testis weight was reduced by more than 50% and sperm reserves declined to less than 20% of control values. By contrast, testis weight and sperm head counts in animals dosed as neonates only decreased to 75% of control values (Table 2).

Table 2. Effect of 37 MBqlkg 114mIn on body and reproductive organ weights and testicular sperm content 63 d postinjection Neonate

Adult Body weight (g) 462 ‘- 22” Treated 518 * 13 Control Testicular weight (g) 0.75 ” 0.02* Treated 1.86 t 0.05 Control Testicular sperm content (X 10B) 0.16 _’ 0.05* Treated 1.02 2 0.04 Control Epididymal weight (g) 0.82 k 0.02* Treated 1.31 r 0.05 Control

40.5 rt 12 421 + 10

Testicular histology Rats dosed with 114mIndisplayed a variety of testicular histologies ranging from a complete absence of spermatogenic activity through varying degrees of normal and abnormal tubular regeneration to normal spermatogenesis. To enable quantification of this testicular damage, tubules were assigned a numerical score between 1 and 5, dependent upon the degree of spermatogenesis present (Table 3). Approximately half of the tubules from adult rats dosed with 114mIn were categorised as grade 1, showing a complete absence of spermatogenic cells and containing only Sertoli cells (Figure If). Conversely only 1% of tubules from animals injected as neonates fell into this category. Almost a further third of the adult tubules were classified as grade 2, defined as early regenerating tubules where only the initial stages of spermatogenesis were present with an even distribution of germ cells around the edge of the tubule (Figure Id). Less than 4% of neonate tubules were in this group. Grade 3 was defined as late regenerating tubules similar to grade 2 but where the germ cell layer was deeper and extended further towards the tubule lumen (Figure lb). Approximately 7 to 15% of adult and neonate tubules were classified in this category. Grade 4 tubules were defined as those tubules that regenerated abnormally. This classification included tubules showing an uneven distribution of regeneration (Figure le) and tubules containing giant cells (Figure lc). The final category, grade 5, was composed of tubules displaying normal spermatogenesis (Figure la). All tubules from control animals fell into this group as did more than 75% of the tubules from animals injected as neonates.

1.43 2 0.10*** 1.85 k 0.04 0.75 k 0.06** 0.99 2 0.04 0.54 * 0.05* 0.93 t 0.02

“Mean f SEM, n = 6. *P < 0.001; **P < 0.01; ***P< 0.05, in comparison with respective control value (unpaired Student’s t test)

neonate rats. At this time, the specific activity of the neonatal tissues was considerably higher than that of the adult tissues. By 63 d, however, the specific activity of the neonate tissues had decreased markedly to levels that were generally below or equal to those of the corresponding adult tissues. The 1*4mIncontent of all adult tissues, with the exception of the spleen, testes, and brain, had also significantly declined by 63 d postinjection. The specific activity of the adult testis actually increased over this time period as a consequence of the decrease in testis weight; however, the whole organ activity remained constant. Body and organ weight and testis sperm head counts No significant changes in body weights were noted between control and treated animals at 63 d postinjection (Table 2). Testis and epididymis weight were reduced in all treated animals as were testicular sperm head counts. The testicular damage was more marked in animals dosed as adults, where

Table 3. Histologic

grading

of testicular

damage

63 d postinjection

of 1i4mIn

% tubules Adult Lesion No spermatogenesis Early regenerating tubule Late regenerating tubule Abnormal regeneration Normal spermatogenesis “Mean 2 SEM, n = 6.

Grade

1 2 3 4 5

Treated 49.5 27.9 9.6 13.0

* 2 ? 2 0

7.9” 2.2 2.19 3.2

Neonate Control 0 0 0 0 100

Treated 0.9 3.15 13.1 4.9 78.2

2 2 + + +

0.1 1.6 1.1 0.47 3.5

Control 0 0 0 0 100

114mIn in adult and neonatal testes

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AL.

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Fig. 1. Spectrum of testicular damage 63 d after treatment of adult rats with 37 MBq/kg ‘14”In. (A) Control tubule exhibiting normal spermatogenesis (M x 200). (B) Late regenerating tubule (x 200). (C) Abnormally regenerating tubule with giant cells (X 200). (D) Early regenerating tubule (x 200). (E) Abnormally regenerating tubule with uneven distribution of spermatogenesis (X 400). (F) Ablated tubule with no spermatogenic activity (X 200).

Isolated tubule experiments

To investigate thle role of transferrin in testicular radionuclide accumulation in more detail, an in vitro model of isolated rat seminiferous tubules was utilised. The methods described for tubule isolation resulted in intact tubules that were impermeable to trypan blue for up to 6 h after isolation.

Both 1r4mInand 125Iwere readily accumulated by tubules from adult (70 d), immature (20 d), and neonatal (7 d) rats. The highest rate of 114mIn accumulation occurred in neonatal tubules, where 9900 + 221 cpm 114mInwere accumulated per mg of tubular protein over the 2-h incubation period, compared to 7252 + 536 cpmlmg and 4288 + 393 cpm/

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transferrin-binding radionuclide, was also examined. As shown in Figure 2b, the addition of up to 200 pg/mL diferric transferrin did not significantly reduce 125Iuptake by tubules from adult, neonatal, or immature rats (control values are 3050 * 173, 5304 ? 27, and 3221 + 290 cpm/mg protein for adult, immature, and neonatal tubules, respectively) . DISCUSSION

0,.,.,.

0

I.,.,.,.,

25

50

pg/ml

01 0

75

iron-saturated

.,.,.,.,.,.,.I., 25 50 75

pg/ml

.I

100

100

iron-saturated

125

150

175

200

trsnsferrin

125

150

175

200

transferrin

Fig. 2. Effect of excess transferrin on the uptake of (A) 114mInand (B) 12sI by isolated seminiferous tubules from adult (0), 20-d-old (O), and 7-d-old (A) rats. Each point represents the mean f SEM from four samples.

mg protein by tubules isolated from immature and adult rats, respectively. The addition of iron-saturated human transferrin to the incubation medium caused a dose-dependent reduction in uptake of *14mTn by tubules from adult and immature rats with maximal inhibition of more than 65% occurring in the presence of 200 PglmL transferrin (Figure 2a). By contrast, 114mInaccumulation by tubules from 7d-old rats in the presence of excess transferrin did not differ significantly from control values. As a negative control, the effect of excess transfer-r-in on tubular accumulation of 1251,a non-

We have previously reported that systemic administration of the 114mInradionuclide to adult rats leads to both cytotoxic and mutagenic damage to testicular germ cells (9). In the current report, we have extended these studies to investigate the role of transferrin in the uptake of 114mInby the seminiferous epithelium and the consequences of testicular radionuclide localisation in adult and neonatal rats. Under normal physiologic conditions, serum transferrin is approximately one-third iron saturated (13). Thus, considerable capacity exists for the binding of systemically administered transferrin-binding radionuclides such as 114mIn.The stability of indium-transferrin is similar to that of irontransferrin (23), and circulating indium-transferrin complexes in trapped blood are likely to make a significant contribution to the levels of i*4mIn observed in most of the resected tissues at 2 d postinjection (Table 1). As transferrin receptors are found on a great many cell types (13,24), however, it is likely that much of the tissue-associated 114mInactivity was specifically associated with cells expressing receptors for transferrin. At 2 d, the specific activity of the neonatal tissues was markedly higher than that of the adult tissues, perhaps reflecting the increased requirement for iron by rapidly proliferating neonatal cells. At 63 d postinjection, only 4 of the resected tissues from the animals injected as adults could be considered to contain significant amounts of 114mIn: the spleen, kidney, testis, and liver (Table 1). This pattern of activity distribution is consistent with u4mIn behaving in a similar manner to iron, as considerable levels of transferrin receptors have been noted in these tissues (13,24). Only the spleen and testes contained a similar portion of the injected activity at 63 d to that at 2 d postinjection. The apparent retention of 114mInby the spleen can be related to this tissue’s role in the destruction of effete red blood cells and the subsequent phagocytosis of *14mInby splenic macrophages. The retention of 114mInby the testes could also be due to phagocytosis of 114mInby resident macrophages in the testic-

114mI~in adult and neonatal testes

ular interstitial tissues. Our previous autoradiohowever, shows a definite graphic evidence, localisation of 114mInwithin seminiferous tubules where it was concentrated within the Sertoli cell cytoplasm and bound to developing germ cells (8). In addition, dominant lethal mutation (DLM) studies show significant induction of DLM after paternal contamination with 114mIn(9). Due to the short range of the radioactive emissions from 114mIn,mutation induction in developing germ cells could only occur if the radionuclide were in extremely close proximity to the target cell. Thus it is unlikely that such mutagenic damaige could be caused by irradiation from outside the tubule. Furthermore the data from the isolated tubule experiments in the current study indicate that seminiferous tubules do accumulate 114mIn.This combined evidence indicates that at least a portion of the testicular 114mInis retained within the seminiferous tubules, suggesting that tubular uptake of *14mIn. is unidirectional. This is consistent with previous observations by Morales and coworkers that transfenin-mediated uptake of 59Fe across the blood-tubule barrier is unidirectional (15). It is interesting to note that 114mInlevels in the adult brain also stayed at a constant, albeit very modest, level. The retention of 114mInby the brain may be considered analogous to the situation in the testis and is probably due to the presence of the blood-brain barrier. Similar to the blood-testis barrier, unidirectional uptake of iron via the receptor-mediated endocytosis of transferrin across the blood-brain barrier has been observed (25). The specific activity of all resected tissues from the animals injected as neonates declined markedly over the experimental period. The extent of this decline can be attributed to a dilution effect caused by the growth of these animals from 20 g at the time of injection to over 400 g at 63 d. However, even when this is accounted for, the whole tissue content of L14mInwas still considerably reduced at 63 d in all resected tissues, particularly the spleen and kidney. The reasons for this are unclear, although the data suggest there may be marked differences in the pharmacokinetic han’dling of transferrin-bound elements between adult and neonatal animals. The amount of 114mInin the testis fell by 50%, which contrasts sharply with the observations from adult animals. This decline may be due to the absence of a competent blood-testis barrier at the time of injection of the radionuclide. At 63 d postinj’ection, *14mIn-treated animals displayed decreased1 testicular and epididymal weights and testicular sperm head counts (Table 2).

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The effects of radiation on the testis are well documented, with most testicular damage arising from the destruction of radiosensitive spermatogonia (26-28). This leads to a sequential decrease in the number of other germ cell types through a maturation depletion process, ultimately causing a reduction in the number of mature sperm and decreased testis and epididymis weight. Testicular damage was more severe in animals dosed as adults than as neonates (Tables 2 and 3). This may be due to a dose-response effect as, whilst all animals were given 37 MBq/kg 114mIn,differences in the pharmacokinetic handling of the radionuclide may reduce the radioactive load to the neonatal testis. Our previous studies (9) have suggested that a minimum threshold dose of 114mInin excess of 2 Gy is required for spermatogonial cell killing in adult rats. As spermatogonia in the immature rat are considered more radioresistant than those in the adult (29), it is probable that an insufficient dose was delivered to the neonatal testis to cause significant destruction of spermatogonia. Furthermore, the absence of the blood-testis barrier in the neonatal animals meant that the 114mInwas not retained within the tubules when the dose was highest. By the time the blood-tubule barrier was fully formed (between 16 and 19 d [17]), these animals would have almost doubled their body weights, thus further diluting the radioactive dose. A considerable spectrum of tubule damage was observed in the treated animals. This damage was more marked in the adult rats where almost half of the tubules exhibited no spermatogenic activity whilst 37% displayed various stages of normal regeneration, with the remainder showing abnormal regeneration. This variety in tubular lesions may be caused by a nonuniform distribution of 114mIn,as it is well recognised that Sertoli cell transferrin receptor expression and transferrin secretion vary with the seminiferous epithelial cycle (30-32). Previous autoradiographic studies with 114mIn-laden testes, however, have revealed a considerable heterogeneity of radionuclide labelling within tubules from histologically comparable stages of the spermatogenic cycle (8). In the in vitro isolated tubule studies, considerably more 114mInwas accumulated by tubules isolated from neonatal and immature rats than by than those from adult rats (Figure 2a). This observation complements data from the in vivo studies showing a higher specific activity in the neonatal testis at 2 d postinjection. The isolated tubule experiments highlighted the role of transferrin in the delivery of 114mInto the testis. Addition of excess diferric trans-

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ferrin to the incubation medium markedly inhibited 114mInuptake by tubules isolated from adult and 20-d rat testes, suggesting competition for tubular transferrin receptors between ii4”In-transferrin and iron-transferrin. By contrast, the addition of excess transferrin did not affect accumulation of 114mInby neonatal (7 d) testes, suggesting that indium accumulation by these tubules is not via the receptormediated endocytosis of transferrin. rz51was used as a negative control in these studies, and, as expected, tubular t2jI accumulation was unaffected by the presence of excess transferrin. In summary, these studies support the contention that transferrin-binding radionuclides such as 114mIncan utilise the physiologic pathway of serum and Sertoli cell transferrin to transcend the bloodtestis barrier and access radiosensitive germ cells in the seminiferous epithelium. Whilst it is difficult to extrapolate directly from rats to humans as human spermatogonia are more radiosensitive than rat spermatogonia and require longer for tubular regeneration after irradiation (33), it should be remembered that the transferrin pathway is ubiquitous amongst mammalian species (13,34). These data suggest that other transfertin-binding radionuchdes may be deposited in the human testes by similar mechanisms. Such testicular radionuclide localisation offers a route for the impairment of spermatogenesis and the transmission of genetic damage via sperm carrying radiation-induced mutations. Acknowledgments - The authors thank Joanne Parker and Anne-Marie Smith for their expert technical assistance. This work is supported by the United Kingdom Co-Ordinating Committee for Cancer Research (Grant No. UKCCCR/RAD90/3) and the Cancer Research Campaign.

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