Cadmium chloride enhances cyclophosphamide-induced cytogenetic damage, testes weight loss and feminization in male mice
Stephanie Blazina, V. Pandurangarao, Scott Smith and Gyula Ficsor
Department of Biological Sciences, Western Michigan University, Kalamazoo, MI.
Ms. Blazina performed the 6 months study part of the experiments. Ms. Blazina also prepared the manuscript in partial fulfillment of the MS degree. As such the manuscript has been reviewed and was approved by her Advisory Committee consisting of Professors Leonard Ginsberg, David Reinhold and Gyula Ficsor (Chair). Data on short-term study was gathered by Dr. Pandurangarao and Scott Smith performed the statistical analysis. Dr. Ficsor has done all the mouse treatments and prepared the photographs in Figure 3.
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Short-(48 h) and long-term ( 6 month) studies were performed to study cadmium chloride=s (CdCl2) ability to enhance the DNA damaging effects of the alkylating agent cyclophosphamide (CP). Groups of four CD1 male mice were injected intraperitoneally (ip) in saline vehicle at 0 h with 0, 0.31, 0.625, 1.25, 2.5, and 5 mg/kg CdCl2 and at 24 h with saline or 50 mg CP/kg.
None of the CdCl2 treatments increased cytogenetic damage in the saline controls in either the short or long-term studies. When 1.25 mg CdCl2/kg or greater doses were followed by CP treatment 24 h later, the frequency of chromatid aberrations (approx. 90% of the damage) and exchanges were significantly increased over CP treatment alone 48 hr after CdCl2 injection. In the long-term study no cytogenetic damage was seen in any of the treated or saline groups. These results indicate that the cadmium enhancement of CP-induced cytogenetic damage seen in the first metaphase after treatment, was not passed on to subsequent cell cycles perhaps owing to apoptosis of the cells suffering extensive DNA damage. We suggest that cadmium enhancement of CP-induced cytogenetic damage in bone marrow cells may have been caused by the replacement of zinc by cadmium in the zinc finger domains of DNA repair enzymes.
In the short-term study 1.25 mg CdCl2/kg or greater doses alone or in combination with CP, caused hemorrhaging and turgidity of the testes. In the long term study testes appeared normal. However, there was a decreasing trend of testes weights. An analysis of variance of the CdCl2, and CdCl2/CP treatment interaction determined that CdCl2 was a significant source of the observed variance (P<0.001).
In the six months study physical examination of the dissected mice at 1.25 mg CdCl2/kg or greater doses alone or with CP caused the appearance of feminine characteristics. This may have been caused by the low level of metallothionen in the testes cells, resulting in an inability to protect Leydig cells from cadmium resulting in destruction of the Leydig cells, the main source of testosterone.
Key words: enhancement of cyclophosphamide-induced cytogenetic damage, cadmium, bone marrow cells, testis damage, feminization, cyclophosphamide
The estimated daily intake of cadmium (Cd) in the U.S.A. by 50-70 kg adult is 50 m g/day . Cadmium is present in food (5-10 μg/kg wet weight), cigarettes (1-2 μg/cigarette) of which 10% enters the body , and in some smelting environments . The half life of cadmium in the human body is 30 years, so under low-level chronic exposure conditions Cd can accumulate in the body . Cadmium=s toxicity is evidenced by its painful effect on bones in the condition known as ITAI-ITAI (ouch-ouch) disease first described in Japan . Other studies have revealed that Cd is responsible for renal tubular dysfunction causing proteinuria and low birth weight in human infants. Others reported that Cd is toxic to the testes [5, 6].
The IACR classified Cd as a probable carcinogen . There is uncertainty about Cd=s clastogenic properties. In a number of reports, including ours, CdCl2 did not cause cytogenetic damage or mutagenic damage in either short or long term studies, while others reported CdCl2 to be a weak chromosome damaging agent [3, 7, 8,].
Cyclophophamide is a nitrogen mustard alkylating agent, as well as a leading anticancer drug and a clastogen . CP acts through alkylation of DNA bases, crosslinking of two DNA strands or by attaching to the triphosphate moiety of the sugar-phosphate DNA backbone. These two reactions of CP with DNA, if unrepaired, may stop the DNA replication machinery leading to double-stranded DNA breaks and hence to chromatid aberrations .
It is of human importance for extrapolation of cytogenetic damage seen in mouse bone marrow cells to determine if the cytogenetic damage seen during the first cell cycle following treatment, will also be seen in the cell lineage of these cells as evaluated in the long term study.
To answer this question a short and long term study was conducted to see if Cd enhances the chromosome damaging effects of CP. This was done by performing a short term (48 h) study and a long term (6 months) study of mouse bone marrow cells in vivo. We also investigated the effects of Cd and CP on the testes by weighing the testes and by visual and physical examination. Our results indicate that CdCl2 enhances the chromosome damaging effect of CP in mouse bone marrow cells seen in the short-term study, but which are not seen 6 months later. In the short-term study, CdCl2 treatment groups of 1.25 mg/kg and greater doses produced hemorrhaging of the testes. Six months later the testes appeared normal in color and texture, but showed a significant decreasing trend of testes weight with CdCl2 as the source of the observed variance (P<0.001). The Cd-treated dissected male mice exhibited feminine characteristics.
MATERIALS AND METHODS
Reagents, mice and injections
CdCl2 was obtained from Aldrich (Milwaukee, WI), CP and colchicine from Sigma (St. Louis, MO). Calcium and magnesium free Hanks Balanced Salt Solution (HBSS) adjusted to a pH of 7.4 with 7.5% sodium bicarbonate were from BRL/Gibco (Grand Island, NY) and Giemsa Stain G-146 powder from Fisher (Fair Lawn, NJ). CD1 male mice were from Charles River (Wilmington, MA).
The mice were acclimated for 1 week prior to treatment, fed and watered ad libitum during acclimation and treatment, weighed before injections and before killing.
Treatment groups consisted of 4 male mice with the exception of the highest 5 mg CdCl2/kg dose in the long-term study when only 2 mice were injected owing to the shortage of same cohort mice available for the study. Female mice were not included because of our interest in the testis response. Doses of CdCl2 ranged from 0.31 mg/kg to 5 mg/kg in doubling intervals as given in Figures 1 and 2 and Tables 1 and 2. Four h before killing all the mice were injected ip with 4 mg/kg colchicine to arrest cells at metaphase and killed 48 h after the saline or Cd injections.
Mice were killed by CO2 narcosis, followed by cervical dislocation and were dissected from the lower abdomen to the upper sternum. Immediately each femur was removed and the tissue was trimmed away with scissors. Excess tissue was detached with Kimwipes. Each end of the femur was pruned until bone marrow was exposed as small red dots. The head of the femur was penetrated with a 23 G needle filled with 3 ml HBSS in a 5 ml syringe warmed to 37oC. The contents of both femurs were flushed with approximately 1.5 ml of HBSS into a 15 ml polypropylene conical centrifuge tube (Fisher, Cat. No. 14-959-49D) pre-marked with the animal=s number. The femur was then inverted and flushed from the other end with the remaining 1.5 ml of HBSS into the test tube.
Immediately the test tubes were centrifuged in a Clay Adams Dynac Centrifuge (Parsippany, NJ) with a 24 x 15 ml angle head at 1000 rpm for 10 min . The supernatant was removed with a Pasteur pipette and rubber bulb, leaving enough liquid (approx. 250 μl) to resuspend the pellet. Next, 7 ml of 0.65% KC1 warmed to 37oC was added to the test tubes and were placed into a 37oC water bath for fifteen min. Test tubes were then centrifuged for 10 min at 1000 rpm.
The supernatant was removed, without disturbing the pellet, leaving approx. 250 μl of 0.65% KCl to resuspend the pellet by tapping the bottom of the tube. Three ml of fresh fixative (3 absolute methanol : 1 glacial acetic acid) were added and the test tube was inverted 3 times, and then was centrifuged for 10 min at 1000 rpm. The supernatant was pipetted off, the pellet resuspended as before and 3 ml of fixative added and stored at 4oC from 1 day to a week until preparation of the slides.
Testes and body morphology observations
In both studies after the femurs were removed, the testes were extracted through an incision made to the lower abdomen. Adhering non-testicular tissue was removed using scissors. They were then weighed, visually inspected. Unaffected testes were light pink in color whereas affected testes were dark red. The testes were physically inspected by rolling them between the thumb and forefinger of a gloved hand. The normal testes felt resilient, whereas those affected by CdCl2 felt turgid to the touch. Following dissection the inside organs of the body were visually examined.
Slide preparation consisted of collecting cells by centrifuging the test tubes at 1000 rpm for 5 min. All but approx. 500 μl of the fixative was removed. The cells were then resuspended. If the cell suspension appeared milky white a few drops of fixative was added back until a slightly turbid cell suspension was obtained.
Grease free microscope slides kept in covered Copeland jars at -20oC for a minium of 1 day. The slides were removed from the Copeland jar and were allowed to collect condensation for approx. 5 sec. Three to four drops of the cell suspension from a Pasteur pipette were immediately placed evenly on each slide. The fixative was then ignited to burst the hypotonically swollen cells and adhere chromosomes and nuclei to the slide. Slides were allowed to dry at room temperature over night and then placed on glass staining rods. Staining consisted of mixing 2 ml of Giesma stock solution  and 1 ml acetone and approximately 23 ml of tap water (very important that top water and not distilled water is used) for a final volume of 25 ml. Each slide was flooded with approx. 2.5 ml stain for 3-5 min. The slides were vigorously rinsed in 1 liter of distilled water, changing the water after every 5 slides. Slides were allowed to air dry in a vertical position overnight and then cover slipped with 22X55 mm coverslips and Permount (Scientific Products, Detroit, MI) and again allowed to dry overnight .
Two or three slides were prepared from each animal depending on the amount of cell suspension available. The slides were marked with the animal number from which the slide was made and by the letters A, B or C to represent the order in which the slides were prepared.
The cytogenetic assay
The animal number on the cover slipped slides was covered with a non-transparent label and assigned a 5 digit random number by a technician unfamiliar with the experiment. The slides from each investigation were boxed in the increasing order of their random number and in the order of their letter designation. The slides were scored following the protocol of Archer et al. .
Fifty to 150 good quality pro-metaphase or metaphase spreads were scored per slide in the short-term and 100 good quality pro-metaphase or metaphase spreads were scored in the long- term study. All slides were scored in the numerical order of their assigned random number which assured randomization of the slides .
In the short term study 34 mice had sufficient number of metaphase spreads on slide A. Slide B was used for 9 and slides A and B for 2 of the mice. One mouse had no scorable metaphases. In the long term study 27 mice had sufficient number of metaphase spreads on slide A. Slide B was used for two and A and B for one and A and C for one mouse. One mouse had no scorable metaphases.
The occurrence of cytogenetic damage was analyzed using Chi-square test, with a continuity correction for the discrete data obtained from the experiment. A 2x2 contingency
table for the control and each treatment group was generated and the Chi-square test performed . Testes weights were analyzed using a two-factor, between-subject experiment. An analysis of variance was performed (ANOVA). The ANOVA for these data was performed by MINITAB Statistical software, Enhanced Version Release 9.1 for VAX/VMS using the general linear model command .
In the short term study CdCl2 doses by themselves produced no cytogenetic damage. Mice injected with saline and 50 mg CP/kg increased cytogenetic damage over saline controls. Doses of 1.25 mg/kg of CdCl2 or higher followed by 50 mg CP/kg 24 h later, produced a significant increase of cytogenetic damage over that was induced by CP treatment alone (Figure1).
Fig. 1. Cadmium chloride doses by themselves did not cause chromatid
aberrations ( red line along X axis). When cadmium chloride treatment was
followed by cyclophosphamide injection 24 hr later, chromatid aberrations were
induced ( ascending blue line). At 1.25 mg cadmium chloride and higher doses,the cadmium enhancing effect is significant.
The short-term bone marrow cytogenetic study in Figure 2 shows that increasing pretreatment doses of CdCl2 with 50 mg CP/kg increased the percentage of abnormal spreads over treatment with CP alone. In addition, Fig. 2 shows that the ratio of spreads with 1-14 chromatid aberrations: >15 chromatid aberrations decreased with increasing doses of CdCl2 treatment. Figure 3 shows photographic examples of chromosome spreads with no, few or many chromatid aberrations as stated in the legends to Figures 3a-i.
Fig.2. Increasing doses of cadmium chloride pretreatment increased the percentage of abnormal spreads over treatment with cyclophosphamide alone (blue and red column parts added). With increasing doses of cadmium chloride, the percentage of metaphase spreads with 1-14 aberrations (blue part of columns) peaked at 2.5 mg/kg, then declined at 5 mg/kg. In contrast, the percentage of cells with >15 aberrations increased (red part of columns) with increasing doses of cadmium chloride.
Of the chromatid aberrations seen in the short-term treatment, approximately 90% were chromatid fragments, and <1% were chromatid breaks and deletions. The remaining were chromatid exchanges (Fig. 3). All photomicrographs were taken at 1,000 magnification.
Fig. 3 a,b. Bone marrow chromosomes from vehicle controls. Cadmium treated chromosome spreads are indistinguishable from vehicle controls (not shown).
Fig 3c. 50 mg Cyclophosphamide/kg. Metaphase with eight fragments and one chromatid break.
3d. 50 mg Cyclophosphamide/kg. Metaphase with exchange
Fig 3e. Injected with 5mg cadmium chloride/kg. 24h later injected with 50mg Cyclophosphamide. Metaphase with approx. 40 fragments and chromatid exchanges.
3f. Injected with 5mg cadmium chloride/kg. 24h later injected with 50mg Cyclophosphamide. Abnormal cells were so frequent, that two highly damaged metaphases were seen in the same view field of the microscope at 1,000X.
Fig 3g. Injected with 5mg Cadmium chloride/kg. 24h later injected with 50mg Cyclophosphamide. Metaphase with approx. 100 fragments
h. Injected with 5mg Cadmium chloride/kg. 24h later injected with 50mg
3i. Injected with 5mg Cadmium chloride/kg. 24h later injected with 50mg
In the long-term study, bone marrow spreads were examined for evidence of cytogenetic damage. Since the mouse karyotype has only telocentric chromosomes, any metacentric chromosome resulting from centric fusion (Robertsonian translocations) would have been readily detected. The many exchanges seen in the short-term study would have been detected as dicentric chromosomes in the long-term study. Rings, fragments or marker chromosomes were also sought. None of these unusual chromosomes or any other kind of cytogenetic damage was seen in the long-term study.
Testes damage and feminization
In the short-term study testis damage was apparent in all the CdCl2 and CdCl2 /CP treatments at 1.25 mg or higher doses of CdCl2. Testis weights were not affected (Table 1 and 2).
In the long term study the testes appeared visually normal. However, there was a decreasing trend of testes weights (Tables 1 and 2). An analysis of variance of the CdCl2, and CdCl2/CP treatment interaction determined that CdCl2 was a significant source of the observed variance (<0.001). Neither the CP nor the CdCl2/CP interaction contributed significantly to the observed variance.
A visual observation of mice exposed to CdCl2 long term (with or without CP) were shown to have fat pads in the hip and buttock area with an overall decrease of muscularity as compared to mice that did not receive CdCl2.
TABLE I. The effect of cadmium chloride and cyclophosphamide (CP) treatments on body and testes weights 48 hours and 6 months after treatment
Forty eight hours treatments
a Died of injection trauma
b Died from scrotal infection from fighting
c CdCl2 was a significant source of variance (P<0.001) and neither CP nor CdCl2/CP interaction contributed significantly to the observed variance.
d Evidence of feminine fat pads.
TABLE 2. Individual body and testes weights and the appearance of testes 48 hours and six months after treatment with cadmium chloride or cyclophosphamide. Table 1 is a summary of these detailed data.
Forty eight hours treatments
Cadmium=s genotoxicity has not been establish unequivocally
The IACR classified Cd as a probable carcinogen .There is uncertainty about Cd=s clastogenic properties. In a number of reports [3, 8, 23] including ours (Figure 1), CdCl2 did not cause cytogenetic damage or mutagenic damage in either the short or long term studies, while others reported CdCl2 to be a weak chromosome damaging agent [3, 7, 8,]. Deaven and Campbell , Gasiorek and Bauchinger , and Mukherjee , found CdCl2 to produce chromosome aberrations at a very low frequency. Murkherjee et al.  found that the majority of damage suffered from CdCl2 were chromatid aberrations.
Cadmium enhances cytogenetic damage by CP and other DNA damaging agents
In the short-term study we found that while Cd alone did not cause cytogenetic damage and CP alone caused a low level of cytogenetic damage in mouse bone marrow cells, when mice were pre-treated with 1.25 mg CdCl2 mg/kg and higher doses followed by CP treatment, the frequency of cytogenetic damage was enhanced over CP. Many cells suffered extensive chromatid damage (Fig. 3e-I).
Yamada also reported that Cd pre-treatment significantly enhanced the chromosome damaging effect of mitomycin C, 4-nitroquinoline 1-oxide, cisplatin, and methyl methanesulfonate in CHO K1 cells. Similar enhancement of mutagenicity of UV light and of three additional mutagens was found in bacteria and mammalian cells in vitro [ 16].
To explain our results we suggest that DNA repair is decreased by the replacement of Zn by Cd in the zinc finger domains of DNA repair enzymes. This idea is supported by the fact that many DNA repair enzymes contain Zn finger motifs and by the similar atomic and chemical properties of Zn and Cd [17, 18].
In our work we have also seen chromatid exchanges resulting from joining of chromatids from different chromosomes. Chromatid exchange requires successful DNA repair of the DNA molecules involved in the exchange. More often than not, chromatid aberrations and exchanges are present in the same cell. Therefore, in a given cell it is not a matter of complete absence of repair but of diminished repair in which some double stranded breaks are successfully repaired leading to chromatid exchanges, and others are not, resulting in chromatid aberrations.
The cytogenetic damage seen in the short-term study was not seen six months later. A possible explanation is apopoptosis of cells which suffered multiple cytogenetic damage.
Cadmium=s effect on the testes observed 48 hours and 6 months after treatment
The damage seen in the testes 48 hr and 6 months after treatment can be explained through the work of Kaur  and Suzuki  who showed that MT is not the major metal binding protein in mammalian testes which may account for the hypersusceptibility of the testes to the necrotic and carcinogenic effects of Cd. Tohyama et al  found that MT was absent in Leydig cells which can lead to decreased testosterone production. Furthermore, Kaur  noted that the major testicular metal binding protein in its native form acts as a Zn reservoir to sustain the biological functions of testicular cells. The inflammation, swelling and hemorrhaging seen 48 hr after Cd-treatment may be due to the diminished quantity and/or absence of MT and by the replacement of Zn by Cd in DNA repair systems.
The decreased testes weight seen 6 months after Cd treatment may have been caused by interstitial hemorrhaging, inflammation seen by us at 48 hr and by Selypes , and selective by chemical ligation reported by Gunn . After 6 months the testes appeared normal visually and to the touch. We believe that the testes were recovering from the initial Cd damage. The comparison of our short and long and testes data agrees with Selypes=  short and long term study in which testes weight did not return to in 6 months.
Feminization of male mice linked with loss of Leydig cells due to cadmium
Six months after Cd treatment of male mice, dissection revealed the presence of subcutaneous estrogen-related fat pads and a decreased musculature as compared to the saline/saline control and the CP alone treated group.
In Bergh=s  investigation of the role of Leydig cells in cadmium toxicity, it was found that a hormonal milieu plays a role in cadmium=s effect on the testes. It was shown that cadmium=s effect on the testes was protected by estrogen treatment, in addition immature testes were insensitive to cadmium treatment.
Although Leydig cells were not analyzed in our short and long term studies, Selypes showed using 1 mg/kg CdCl2 that Leydig cell death occurred in a 3 day study. We suggest that the physical changes we observed in body morphology may be due to death of Leydig cells, which lead to a decrease of testosterone, and subsequent increase of the estrogen level.
The short term study of bone marrow cells showed that CdCl2 treatment prior to CP treatment enhanced the DNA damaging effect of CP seen as chromatid damage in a dose-dependent manner. But after six months a similar enhancing effect was absent.
We suggest that the extensive Cd-induced increase in CP-induced DNA damage seen after 48 h was caused by the replacement of Zn by Cd in the Zinc finger motifs of repair enzymes. This DNA damage may have caused extensive apoptosis so that the enhancing effect seen 48 hours after treatment, was not seen after six months.
Forty eight hours after treatment cadmium alone or with CP caused inflammation and hemorrhaging of the testes. After six months testes appeared normal in appearance but testes weights were decreased.
Cd treatment also caused feminization of male mice probably owing to loss of Leydig cells and decreased testosterone.
We would like to thank K. Block for consultation and M. Lincoln, T. Nyirenda, R. Behrje, A. Sandor, for technical assistance. Sridhar Kaundinya helped to prepare the manuscript for the internet. Financial support was provided from the Center for the Study of Environmental Signal Transmission of Western Michigan University, and S.Fry M.D., Minneapolis, MN.
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