Hydroxylamine moiety of developmental toxicants is associated with early cell death: a structure-activity analysis

BACKGROUND: Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. METHODS: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. RESULTS: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. CONCLUSIONS: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.     

CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

The induction of transient increases in mitotic rate in murine tissues following prolonged intravenous infusions of hydroxyurea.

Intravenous infusions of hydroxyurea were established in mice and maintained for periods up to 48 hr. The influence of different rates of hydroxyurea infusion on the number of viable cells gathered in S phase was studied in eight different mouse tissues. An infusion rate which was sufficiently slow not to block thymidine incorporation completely, resulted in gathering of cells in S phase while offering some protection against hydroxyurea-induced cell death. The duration of the period of DNA synthesis following release from hydroxyurea inhibition appeared to be shortened in some tissues. After the release of hydroxyurea blockades maintained for 12-24 hr, each of the tissues showed sharp increases in mitotic activity and peak mitotic index values were as much as twenty times greater than values found in tissues of control animals. An important finding was that the time of maximal mitotic activity for different tissues after release of blockade could differ by many hours.     

The cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D0, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D0, 0.43 Gy) than the other phases of the cell cycle (D0, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.     

THE INDUCTION OF TRANSIENT INCREASES IN MITOTIC RATE IN MURINE TISSUES FOLLOWING PROLONGED INTRAVENOUS INFUSIONS OF HYDROXYUREA

Intravenous infusions of hydroxyurea were established in mice and maintained for periods up to 48 hr. The influence of different rates of hydroxyurea infusion on the number of viable cells gathered in S phase was studied in eight different mouse tissues. An infusion rate which was sufficiently slow not to block thymidine incorporation completely, resulted in gathering of cells in S phase while offering some protection against hydroxyurea-induced cell death. The duration of the period of DNA synthesis following release from hydroxyurea inhibition appeared to be shortened in some tissues. After the release of hydroxyurea blockades maintained for 12–24 hr, each of the tissues showed sharp increases in mitotic activity and peak mitotic index values were as much as twenty times greater than values found in tissues of control animals. An important finding was that the time of maximal mitotic activity for different tissues after release of blockade could differ by many hours.     

The Cell Cycle of Spermatogonial Colony Forming Stem Cells In the Cba Mouse After Neutron Irradiation

Abstract. In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D_o, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D_o, 0.43 Gy) than the other phases of the cell cycle (D_o, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. the data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.     

Cytodynamics in the thymus of young adult mice: a quantitative study on the loss of thymic blast cells and non-proliferative small thymocytes.

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse 3H-TdR, and (2) nigrosin-dye exclusion combined with 3H-TdR-autoradiography. It was calculated that about 17 percent of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2-5 percent/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after 3H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of 3H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated 3H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1-6 percent/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0-02 percent/hr and 0-0006 percent/hr respectively. Cell loss due to disintegration was less than 2 percent of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 percent of the small viable thymocytes re-enter the generative cell pool, whereas about 55 percent disappear by emigration.     

Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues

Hydroxyurea (HU), a ribonucleotide reductase inhibitor, induces morphological anomalies in the central nervous system (CNS), craniofacial tissues and limb buds in animals, and neonatal respiratory distress in humans. In the present study, pregnant mice were treated with 400 mg/kg of HU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue to clarify the mechanisms of HU-induced fetotoxicity and teratogenecity. At 6 and 12 HAT, a moderate to marked increase in the number of pyknotic cells was detected in the CNS and lung. A mild increase in the number of pyknotic cells was also found in the craniofacial mesenchymal tissues, limb buds and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results suggest that the HU-induced fetotoxicity is characterized by excess apoptotic cell death in the fetal tissues, and that such excess cell death in the fetal CNS, lung, craniofacial tissue and limb bud may have a certain relation to the later occurrence of morphological or functional anomalies reported in these tissues following HU-administration.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE II. MAMMARY TUMOR CELL KINETICS

The cellular kinetics of C3H mouse mammary tumors were studied following a single dose (3 mg/g body weight) of hydroxyurea (HU). This dose was large enough to cause a significant perturbation in the growth curves of these tumours. This was accomplished by labeling the cells with tritiated 5-iodo-2'-deoxyuridine and performing detailed autoradiographic analysis. This dose of HU caused a temporary inhibition in growth and completely inhibited DNA synthesis for 4–5 hr. The HU-killed cells (pyknotic and karyorrhectic) reach a maximum around 10–12 hr and are apparently all removed in about 1 day. Tumors from a fast-growing line (S102F) showed some evidence for cell synchrony upon recovery from HU inhibition but desynchronization occurred within one cell cycle. The cell generation time was not decreased during the acute recovery phase, but the growth fraction shifted from 0·6 to 1·0, and the data suggested that the normal flow of cells from the proliferating pool to the degenerate pool was temporarily interrupted. The cellular kinetic parameters have probably returned to normal by 48 hr after the HU injection.     

Analysis of the effect of hydroxyurea on stem cell (CFU-s) kinetics

Abstract. Hydroxyurea induces profound changes in the pluripotential haemopoietic stem cell (CFU-s) kinetics. The main feature of these changes is a synchronous entry of resting Go CFU-s into the cell cycle. The analysis of the passage of the CFU-s cohort through the cell cycle has been largely based on the examination of the fraction of CFU-s which synthesize DNA in the S phase of the cell cycle. This analysis has, however, been hampered by the fact that both the sensitivity of the S phase CFU-s to hydroxyurea and their sensitivity in the [³H] thymidine suicide technique vary as the cells pass through the S phase. Methods which overcome these difficulties have been used in the experiments presented in this paper.
It was demonstrated that hydroxyurea kills only about 80% of the S phase CFU-s. The sensitivity to hydroxyurea gradually decreases as the cells approach the middle part of the S phase and increases again as the cells enter the late portions of the S phase.
The degree of CFU-s synchrony at the point of entry into and exit from, the S phase has been established. Mathematical analysis of the available data suggests that CFU-s pass through the S phase with a mean transit time of 4–79 hr (standard deviation, 1.45 hr).
Hydroxyurea, administered in vivo , blocks CFU-s in the late G1 phase. The duration of this G1-S block, induced by a dose of 1000 mg of hydroxyurea per kg body weight, is approximately 2 hr. The CFU-s in the middle of the S phase, which survive hydroxyurea administration, are also blocked in their passage through the S phase. These cells, however, seem to finish the S phase with a delay of approximately 2 hr.     

Glutamate-Treated Rat Cortical Neuronal Cultures Die in a Way Different from the Classical Apoptosis Induced by Staurosporine

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized byin situend-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.     

Developmental retinal ganglion cell death and retinotopicity of the murine retinocollicular projection

During mammalian visual system development, retinal ganglion cells (RGCs) undergo extensive apoptotic death. In mouse retina, approximately 50% of RGCs present at birth (postnatal day 0; P0) die by P5, at a time when axons innervate central targets such as the superior colliculus (SC). We examined whether RGCs that make short‐range axonal targeting errors within the contralateral SC are more likely to be eliminated during the peak period of RGC death (P1‐P5), compared with RGCs initially making more accurate retinotopic connections. A small volume (2.3 nL) of the retrograde nucleophilic dye Hoechst 33342 was injected into the superficial left SC of anesthetized neonatal C57Bl/6J mice at P1 (n = 5) or P4 (n = 8), and the contralateral retina wholemounted 12 hr later. Retrogradely labelled healthy and dying (pyknotic) RGCs were identified by morphological criteria and counted. The percentage of pyknotic RGCs was analyzed in relation to distance from the area of highest density RGC labelling, presumed to represent the most topographically accurate population. As expected, pyknotic RGC density at P1 was significantly greater than P4 (p < 0.05). At P4, the density of healthy RGCs 500–750 µm away from the central region was significantly less, although this was not reflected in altered pyknotic rates. However, at P1 there was a trend (p = 0.08) for an increased proportion of pyknotic RGCs, specifically in temporal parts of the retina outside the densely labelled center. Overall, the lack of consistent association between short‐range targeting errors and cell death suggests that most postnatal RGC loss is not directly related to topographic accuracy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 51–60, 2018     

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.     

Stem cell (CFUs) proliferation inhibitor in blood of mice injected with hydroxyurea

Abstract. Analysis of the mouse haemopoietic stem cell (CFUs) kinetics after hydroxyurea administration has provided an in vivo assay suitable for detection of factors which inhibit recruitment of non-proliferating G₀-CFUs into cell cycle, or transit of CFU's through the G₁ phase. Using this assay, it has been demonstrated that plasma obtained from mice which had received hydroxyurea approximately 12–14 hr previously, possesses a factor which inhibited the triggering of CFUs into the cell cycle. The appearance of this CFUs proliferation inhibitor occurred at a time when 60–70% of the CFUs were synchronized in the S phase of the cell cycle, as a consequence of hydroxyurea action. Some basic properties of the inhibitor were investigated.     

Comparative analysis of the destroying effects of prednisolone and irradiation of lymphoid cells]

The hormone-induced and post-irradiation changes in the molecular weight of a single-stranded DNA (SSDNA) in alkaline nuclear lysates and the activities of DNAses and pyknotic nuclei from rat thymocytes were studied. It was shown that 1 hr after injection of prednisolone (1 mg per 100 g of body weight) the molecular weight of SSDNA in the lymphoid organs is decreased with a subsequent increase by the 6th hour. The hormone-induced degradation of DNA is not accompanied by any marked increase in the activities of DNAses or by an appearance of pykotic nuclei in the thymocytes. The irradiation of the animals at a dose of 900 R leads to an irreversible decrease of the molecular weight of SSDNA in the lymphoid organs, to a steady increase of the DNAse activity and a sharp increase of the amount of pyknotic nuclei in the thymocytes. Studies on the mechanism of post-hormonal degradation of DNA in rat thymocytes in vitro demonstrated that prednisolone exerts its effects on the early and late stages of DNA degradation.     

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.     

Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture

Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo. Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures. In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes. In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine. The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos. The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads. In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M. After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%). Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation. The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.     

EFFECTS OF HYDROXYUREA ON THE KINETICS OF COLONY FORMING UNITS OF BONE MARROW IN THE MOUSE

The number of nucleated bone marrow cells, the number of CFU and the number of DNA-synthesizing cells in the mouse were studied after injection of hydroxyurea. It was found that one injection provokes a partial synchronization of surviving cells and probably stimulates the transition of CFU from the quiescent to the cycling state. the changes of the proportion of CFU in the S phase makes it possible to estimate approximately a cell cycle duration of about 12 hr.     

Analysis of growth of tetraploid nuclei in roots of Vicia faba

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.